Recombinational cloning using nucleic acids having recombination sites

ABSTRACT

Recombinational cloning is provided by the use of nucleic acids, vectors and methods, in vitro and in vivo, for moving or exchanging segments of DNA molecules using engineered recombination sites and recombination proteins to provide chimeric DNA molecules that have the desired characteristic(s) and/or DNA segment(s).

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. applicationSer. No. 10/300,892, filed Nov. 21, 2002, which is divisional of U.S.application Ser. No. 09/907,719, filed Jul. 19, 2001, which is adivisional of U.S. application Ser. No. 09/177,387, filed Oct. 23, 1998(now abandoned), which claims the benefit of the filing date of U.S.Provisional Application No. 60/065,930, filed Oct. 24, 1997, thedisclosures of which are incorporated by reference herein in theirentireties. The present application is also related to U.S. applicationSer. No. 08/663,002, filed Jun. 7, 1996 (now U.S. Pat. No. 5,888,732),which is a continuation-in-part of U.S. application Ser. No. 08/486,139,filed Jun. 7, 1995 (now abandoned), the disclosures of whichapplications are incorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to recombinant DNA technology. DNAand vectors having engineered recombination sites are provided for usein a recombinational cloning method that enables efficient and specificrecombination of DNA segments using recombination proteins. The DNAs,vectors and methods are useful for a variety of DNA exchanges, such assubcloning of DNA, in vitro or in vivo.

[0004] 2. Related Art

[0005] Site-specific recombinases. Site-specific recombinases areproteins that are present in many organisms (e.g. viruses and bacteria)and have been characterized to have both endonuclease and ligaseproperties. These recombinases (along with associated proteins in somecases) recognize specific sequences of bases in DNA and exchange the DNAsegments flanking those segments. The recombinases and associatedproteins are collectively referred to as “recombination proteins” (see,e.g.,, Landy, A., Current Opinion in Biotechnology 3:699-707 (1993)).

[0006] Numerous recombination systems from various organisms have beendescribed. See, e.g., Hoess et al., Nucleic Acids Research 14(6):2287(1986); Abremski et al., J. Biol. Chem.261(1):391 (1986); Campbell, J.Bacteriol. 174(23):7495 (1992); Qian et al., J. Biol. Chem. 267(11):7794(1992); Araki et al., J. Mol. Biol. 225(1):25 (1992); Maeser andKahnmann Mol. Gen. Genet. 230:170-176) (1991); Esposito et al., Nucl.Acids Res. 25(18):3605 (1997).

[0007] Many of these belong to the integrase family of recombinases(Argos et al. EMBO J. 5:433-440 (1986)). Perhaps the best studied ofthese are the Integrase/att system from bacteriophage λ (Landy, A.Current Opinions in Genetics and Devel. 3:699-707 (1993)), the Cre/loxPsystem from bacteriophage P1 (Hoess and Abremski (1990) In Nucleic Acidsand Molecular Biology, vol. 4. Eds.: Eckstein and Lilley,Berlin-Heidelberg: Springer-Verlag; pp. 90-109), and the FLP/FRT systemfrom the Saccharomyces cerevisiae 2μ circle plasmid (Broach et al. Cell29:227-234 (1982)).

[0008] Backman (U.S. Pat. No. 4,673,640) discloses the in vivo use of λrecombinase to recombine a protein producing DNA segment by enzymaticsite-specific recombination using wild-type recombination sites attB andattP.

[0009] Hasan and Szybalski (Gene 56:145-151 (1987)) discloses the use ofλ Int recombinase in vivo for intramolecular recombination between wildtype attP and attB sites which flank a promoter. Because theorientations of these sites are inverted relative to each other, thiscauses an irreversible flipping of the promoter region relative to thegene of interest.

[0010] Palazzolo et al. Gene 88:25-36 (1990), discloses phage lambdavectors having bacteriophage λ arms that contain restriction sitespositioned outside a cloned DNA sequence and between wild-type loxPsites. Infection of E. coli cells that express the Cre recombinase withthese phage vectors results in recombination between the loxP sites andthe in vivo excision of the plasmid replicon, including the cloned cDNA.

[0011] Pósfai et al. (Nucl. Acids Res. 22:2392-2398 (1994)) discloses amethod for inserting into genomic DNA partial expression vectors havinga selectable marker, flanked by two wild-type FRT recognition sequences.FLP site-specific recombinase as present in the cells is used tointegrate the vectors into the genome at predetermined sites. Underconditions where the replicon is functional, this cloned genomic DNA canbe amplified.

[0012] Bebee et al. (U.S. Pat. No. 5,434,066) discloses the use ofsite-specific recombinases such as Cre for DNA containing two loxP sitesis used for in vivo recombination between the sites.

[0013] Boyd (Nucl. Acids Res. 21:817-821 (1993)) discloses a method tofacilitate the cloning of blunt-ended DNA using conditions thatencourage intermolecular ligation to a dephosphorylated vector thatcontains a wild-type loxP site acted upon by a Cre site-specificrecombinase present in E. coli host cells.

[0014] Waterhouse et al. (PCT No. 93/19172 and Nucleic Acids Res. 21(9):2265 (1993)) disclose an in vivo method where light and heavy chainsof a particular antibody were cloned in different phage vectors betweenloxP and loxP 511 sites and used to transfect new E. coli cells. Cre,acting in the host cells on the two parental molecules (one plasmid, onephage), produced four products in equilibrium: two differentcointegrates (produced by recombination at either loxP or loxP 511sites), and two daughter molecules, one of which was the desiredproduct.

[0015] In contrast to the other related art, Schlake & Bode(Biochemistry 33:12746-12751 (1994)) discloses an in vivo method toexchange expression cassettes at defined chromosomal locations, eachflanked by a wild type and a spacer-mutated FRT recombination site. Adouble-reciprocal crossover was mediated in cultured mammalian cells byusing this FLP/FRT system for site-specific recombination.

[0016] Transposases. The family of enzymes, the transposases, has alsobeen used to transfer genetic information between replicons. Transposonsare structurally variable, being described as simple or compound, buttypically encode the recombinase gene flanked by DNA sequences organizedin inverted orientations. Integration of transposons can be random orhighly specific. Representatives such as Tn7, which are highlysite-specific, have been applied to the in vivo movement of DNA segmentsbetween replicons (Lucklow et al., J. Virol. 67:4566-4579 (1993)).

[0017] Devine and Boeke Nucl. Acids Res. 22:3765-3772 (1994), disclosesthe construction of artificial transposons for the insertion of DNAsegments, in vitro, into recipient DNA molecules. The system makes useof the integrase of yeast TY1 virus-like particles. The DNA segment ofinterest is cloned, using standard methods, between the ends of thetransposon-like element TY1. In the presence of the TY1 integrase, theresulting element integrates randomly into a second target DNA molecule.

[0018] DNA cloning.The cloning of DNA segments currently occurs as adaily routine in many research labs and as a prerequisite step in manygenetic analyses. The purpose of these clonings is various, however, twogeneral purposes can be considered: (1) the initial cloning of DNA fromlarge DNA or RNA segments (chromosomes, YACs, PCR fragments, mRNA,etc.), done in a relative handful of known vectors such as pUC, pGem,pBlueScript, and (2) the subcloning of these DNA segments intospecialized vectors for functional analysis. A great deal of time andeffort is expended both in the transfer of DNA segments from the initialcloning vectors to the more specialized vectors. This transfer is calledsubcloning.

[0019] The basic methods for cloning have been known for many years andhave changed little during that time. A typical cloning protocol is asfollows:

[0020] (1) digest the DNA of interest with one or two restrictionenzymes;

[0021] (2) gel purify the DNA segment of interest when known;

[0022] (3) prepare the vector by cutting with appropriate restrictionenzymes, treating with alkaline phosphatase, gel purify etc., asappropriate;

[0023] (4) ligate the DNA segment to the vector, with appropriatecontrols to eliminate background of uncut and self-ligated vector;

[0024] (5) introduce the resulting vector into an E. coli host cell;

[0025] (6) pick selected colonies and grow small cultures overnight;

[0026] (7) make DNA minipreps; and

[0027] (8) analyze the isolated plasmid on agarose gels (often afterdiagnostic restriction enzyme digestions) or by PCR.

[0028] The specialized vectors used for subcloning DNA segments arefunctionally diverse. These include but are not limited to: vectors forexpressing genes in various organisms; for regulating gene expression;for providing tags to aid in protein purification or to allow trackingof proteins in cells; for modifying the cloned DNA segment (e.g.,generating deletions); for the synthesis of probes (e.g., riboprobes);for the preparation of templates for DNA sequencing; for theidentification of protein coding regions; for the fusion of variousprotein-coding regions; to provide large amounts of the DNA of interest,etc. It is common that a particular investigation will involvesubcloning the DNA segment of interest into several differentspecialized vectors.

[0029] As known in the art, simple subclonings can be done in one day(e.g., the DNA segment is not large and the restriction sites arecompatible with those of the subcloning vector). However, many othersubclonings can take several weeks, especially those involving unknownsequences, long fragments, toxic genes, unsuitable placement ofrestriction sites, high backgrounds, impure enzymes, etc. Subcloning DNAfragments is thus often viewed as a chore to be done as few times aspossible. Several methods for facilitating the cloning of DNA segmentshave been described, e.g., as in the following references.

[0030] Ferguson, J., et al. Gene 16:191 (1981), discloses a family ofvectors for subcloning fragments of yeast DNA. The vectors encodekanamycin resistance. Clones of longer yeast DNA segments can bepartially digested and ligated into the subcloning vectors. If theoriginal cloning vector conveys resistance to ampicillin, nopurification is necessary prior to transformation, since the selectionwill be for kanamycin.

[0031] Hashimoto-Gotoh, T., et al. Gene 41:125 (1986), discloses asubcloning vector with unique cloning sites within a streptomycinsensitivity gene; in a streptomycin-resistant host, only plasmids withinserts or deletions in the dominant sensitivity gene will survivestreptomycin selection.

[0032] Accordingly, traditional subcloning methods, using restrictionenzymes and ligase, are time consuming and relatively unreliable.Considerable labor is expended, and if two or more days later thedesired subclone can not be found among the candidate plasmids, theentire process must then be repeated with alternative conditionsattempted. Although site specific recombinases have been used torecombine DNA in vivo, the successful use of such enzymes in vitro wasexpected to suffer from several problems. For example, the sitespecificities and efficiencies were expected to differ in vitro;topologically-linked products were expected; and the topology of the DNAsubstrates and recombination proteins was expected to differsignificantly in vitro (see, e.g., Adams et al, J. Mol. Biol. 226:661-73(1992)). Reactions that could go on for many hours in vivo were expectedto occur in significantly less time in vitro before the enzymes becameinactive. Multiple DNA recombination products were expected in thebiological host used, resulting in unsatisfactory reliability,specificity or efficiency of subcloning. Thus, in vitro recombinationreactions were not expected to be sufficiently efficient to yield thedesired levels of product.

[0033] Accordingly, there is a long felt need to provide an alternativesubcloning system that provides advantages over the known use ofrestriction enzymes and ligases.

SUMMARY OF THE INVENTION

[0034] The present invention provides nucleic acids, vectors and methodsfor obtaining amplified, chimeric or recombinant nucleic acid moleculesusing recombination proteins and at least one recombination site, invitro or in vivo. These methods are highly specific, rapid, and lesslabor intensive than standard cloning or subcloning techniques. Theimproved specificity, speed and yields of the present inventionfacilitates DNA or RNA cloning or subcloning, regulation or exchangeuseful for any related purpose.

[0035] The present invention relates to nucleic acids, vectors andmethods for moving or exchanging nucleic acid segments (preferably DNAsegments or fragments) using at least one recombination site and atleast one recombination protein to provide chimeric DNA molecules whichhave the desired characteristic(s) and/or DNA segment(s). Use of theinvention thus allows for cloning or subcloning such nucleic acidmolecules into a variety of vectors. Generally, one or more parentnucleic acid molecules (preferably DNA molecules) are recombined to giveone or more daughter molecules, at least one of which is the desiredProduct molecule, which is preferably a vector comprising the desirednucleic acid segment. The invention thus relates to nucleic acidmolecules, vectors and methods to effect the exchange and/or to selectfor one or more desired products.

[0036] One embodiment of the present invention relates to a method ofmaking chimeric molecule, which comprises

[0037] (a) combining in vitro or in vivo

[0038] (i) one or more Insert Donor molecules comprising a desirednucleic acid segment flanked by a first recombination site and a secondrecombination site, wherein the first and second recombination sites donot substantially recombine with each other;

[0039] (ii) one or more Vector Donor molecules comprising a thirdrecombination site and a fourth recombination site, wherein the thirdand fourth recombination sites do not substantially recombine with eachother; and

[0040] (iii) one or more site specific recombination proteins capable ofrecombining the first and third recombinational sites and/or the secondand fourth recombinational sites;

[0041] thereby allowing recombination to occur, so as to produce atleast one cointegrate nucleic acid molecule, at least one desiredProduct nucleic acid molecule which comprises said desired segment, andoptionally a Byproduct nucleic acid molecule; and then, optionally,

[0042] (b) selecting for the Product or Byproduct DNA molecule.

[0043] In another embodiment, the present invention relates to a methodof making chimeric molecule, which comprises

[0044] (a) combining in vitro or in vivo

[0045] (i) one or more Insert Donor molecules comprising a desirednucleic acid segment flanked by two or more recombination sites whereinsaid recombination sites do not substantially recombine with each other;

[0046] (ii) one or more Vector Donor molecules comprising two or morerecombination sites, wherein said recombination sites do notsubstantially recombine with each other; and

[0047] (iii) one or more site specific recombination proteins;

[0048] (b) incubating said combination under conditions sufficient totransfer one or more said desired segments into one or more of saidVector Donor molecules, thereby producing one or more Product molecules.The resulting Product molecules may optionally be selected or isolatedaway from other molecules such as cointegrate molecules, Byproductmolecules, and unreacted Vector Donor molecules or Insert Donormolecules. In a preferred aspect of the invention, the Insert Donormolecules are combined with one or more different Vector Donormolecules, thereby allowing for the production of different Productmolecules in which the nucleic acid of interest is transferred into anynumber of different vectors in the single step.

[0049] In accordance with the invention, the above methods may bereversed to provide the original Insert Donor molecules which may thenbe used in combination with one or more different Vector Donor moleculesto produce new Product or Byproduct molecules. Alternatively, theProduct molecules produced by the method of the invention may serve asthe Insert Donor molecules which may be used directly in combinationwith one or more different Vector Donor molecules, thereby producing newProduct or Byproduct molecules. Thus, nucleic acid molecules of interestmay be transferred or moved to any number of desired vectors, therebyproviding an efficient means for subcloning molecules of interest.

[0050] Thus, the invention relates to combining a Product molecule witha second Vector Donor molecules to produce a second Product molecule.The second Product DNA molecule may then be utilized in combination witha third Vector Donor molecule to produce a third Product molecule. Thisprocess of the invention may be repeated any number of times to transferor move the insert of interest into any number of different vectors. Inthis aspect of the invention, a combination of two or more differentVector Donor molecules may be combined with the Product molecule toproduce in a single step different Product molecules in which thedesired nucleic acid segment (derived from the Product DNA molecule) istransferred into any number of different vectors.

[0051] In particular, the present invention relates to a method forcloning or subcloning one or more desired nucleic acid moleculescomprising

[0052] (a) combining in vitro or in vivo

[0053] (i) one or more Insert Donor molecules comprising one or moredesired nucleic acid segments flanked by at least two recombinationsites, wherein said recombination sites do not substantially recombinedwith each other;

[0054] (ii) one or more Vector Donor molecules comprising at least tworecombination sites, wherein said recombination sites do notsubstantially recombine with each other; and

[0055] (iii) one or more site specific recombination proteins;

[0056] (b) incubating said combination under conditions sufficient toallow one or more of said desired segments to be transferred into one ormore of said Vector Donor molecules, thereby producing one or moreProduct molecules;

[0057] (c) optionally selecting for or isolating said Product molecule;

[0058] (d) combining in vitro or in vivo

[0059] (i) one or more of said Product molecules comprising said desiredsegments flanked by two or more recombination sites, wherein saidrecombination sites do not substantially recombine with each other;

[0060] (ii) one or more different Vector Donor molecules comprising twoor more recombination sites, wherein said recombination sites do notsubstantially recombine with each other; and

[0061] (iii) one or more site specific recombination protein; and

[0062] (e) incubating said combination under conditions sufficient totransfer one or more of said desired segments into one or more of saiddifferent Vector Donor molecules, thereby producing one or moredifferent Product molecules.

[0063] In accordance with the invention, Vector Donor molecules maycomprise vectors which may function in a variety of systems or hostcells. Preferred vectors for use in the invention include prokaryoticvectors, eukaryotic vectors or vectors which may shuttle between variousprokaryotic and/or eukaryotic systems (e.g. shuttle vectors). Preferredprokaryotic vectors for use in the invention include but are not limitedto vectors which may propagate and/or replicate in gram negative and/orgram positive bacteria, including bacteria of the genus Escherichia,Salmonella, Proteus, Clostridium, Klebsiella, Bacillus, Streptomyces,and Pseudomonas and preferably in the species E. coli. Eukaryoticvectors for use in the invention include vectors which propagate and/orreplicate and yeast cells, plant cells, mammalian cells, (particularlyhuman), fungal cells, insect cells, fish cells and the like. Particularvectors of interest include but are not limited to cloning vectors,sequencing vectors, expression vectors, fusion vectors, two-hybridvectors, gene therapy vectors, and reverse two-hybrid vectors. Suchvectors may be used in prokaryotic and/or eukaryotic systems dependingon the particular vector.

[0064] The Insert Donor molecules used in accordance with the inventionpreferably comprise two or more recombination sites which allow theinsert (e.g. the nucleic acid segment of interest) of the Donormolecules to be transferred or moved into one or more Vector Donormolecules in accordance with the invention. The Insert Donor moleculesof the invention may be prepared by any number of techniques by whichtwo or more recombination sites are added to the molecule of interest.Such means for including recombination sites to prepare the Insert Donormolecules of the invention includes mutation of a nucleic acid molecule(e.g. random or site specific mutagenesis), recombinant techniques (e.g.ligation of adapters or nucleic acid molecules comprising recombinationsites to linear molecules), amplification (e.g. using primers whichcomprise recombination sites or portions thereof) transposition (e.g.using transposons which comprise recombination sites), recombination(e.g. using one or more homologous sequences comprising recombinationsites), nucleic acid synthesis (e.g. chemical synthesis of moleculescomprising recombination sites or enzymatic synthesis using variouspolymerases or reverse transcriptases) and the like. In accordance withthe invention, nucleic acid molecules to which one or more recombinationsites are added may be any nucleic acid molecule derived from any sourceand may include non naturally occurring nucleic acids (e.g. RNA's; seeU.S. Pat. Nos. 5,539,082 and 5,482,836). Particularly preferred nucleicacid molecules are DNA molecules (single stranded or double stranded).Additionally, the nucleic acid molecules of interest for producingInsert Donor molecules may be linear or circular and further maycomprise a particular sequence of interest (e.g. a gene) or may be apopulation of molecules (e.g. molecules generated from a genomic or cDNAlibraries).

[0065] Thus, the invention relates to a number of methods for preparingInsert Donor molecules and the Insert Donor molecules produced by suchmethods. In one aspect of the invention, primers comprising one or morerecombination sites or portions thereof are used in the nucleic acidsynthesis or nucleic acid amplification to prepare the Insert Donormolecules of the invention. Thus, the invention relates to a method ofsynthesizing a nucleic acid molecule comprising:

[0066] (a) mixing one or more nucleic acid templates with a polypeptidehaving polymerase activity and one or more primers comprising one ormore recombination sites or portions thereof; and

[0067] (b) incubating said mixture under conditions sufficient tosynthesize one or more nucleic acid molecules which are complementary toall or a portion of said templates and which comprises one or morerecombination sites. In accordance with the invention, the synthesizednucleic acid molecule comprising one or more recombination sites may beused as templates under appropriate conditions to synthesize nucleicacid molecules complementary to all or a portion of the recombinationsite containing templates, thereby forming double stranded moleculescomprising one or more recombination sites. Preferably, such secondsynthesis step is performed in the presence of one or more primerscomprising one or more recombination sites. In yet another aspect, thesynthesized double stranded molecules may be amplified using primerswhich may comprise one or more recombination sites.

[0068] In another aspect of the invention, one or more recombinationsites may be added to nucleic acid molecules by any of a number ofnucleic acid amplification techniques. In particular, such methodcomprises:

[0069] (a) contacting a first nucleic acid molecule with a first primermolecule which is complementary to a portion of said first nucleic acidmolecule and a second nucleic acid molecule with a second primermolecule which is complementary to a portion of said second nucleic acidmolecule in the presence of one or more polypeptides having polymerasesactivity;

[0070] (b) incubating said molecules under conditions sufficient to forma third nucleic acid molecule complementary to all or a portion of saidfirst nucleic acid molecule and the fourth nucleic acid moleculecomplementary to all or a portion of said second nucleic acid molecule;

[0071] (c) denaturing said first and third and said second and fourthnucleic acid molecules; and

[0072] (d) repeating steps (a) through (c) one or more times,

[0073] wherein said first and/or said second primer molecules compriseone or more recombination sites or portions thereof.

[0074] In yet another aspect of the invention, a method for adding oneor more recombination sites to nucleic acid molecules may comprise:

[0075] (a) contacting one or more nucleic acid molecules with one ormore adapters or nucleic acid molecules which comprise one or morerecombination sites or portions thereof; and

[0076] (b) incubating said mixture under conditions sufficient to addone or more recombination sites to said nucleic acid molecules.Preferably, linear molecules are used for adding such adapters ormolecules in accordance with the invention and such adapters ormolecules are preferably added to one or more termini of such linearmolecules. The linear molecules may be prepared by any techniqueincluding mechanical (e.g. sonication or shearing) or enzymatic (e.g.nucleases such as restriction endonucleases). Thus, the method of theinvention may further comprise digesting the nucleic acid molecule withone or more nucleases (preferably any restriction endonucleases) andligating one or more of the recombination site containing adapters ormolecules to the molecule of interest. Ligation may be accomplishedusing blunt ended or stick ended molecules. Alternatively,topoisomerases may be used to introduce recombination sites inaccordance with the invention. Topoisomerases cleave and rejoin nucleicacid molecules and therefore may be used in place of nucleases andligases.

[0077] In another aspect, one or more recombination sites may be addedto nucleic acid molecules by de novo synthesis. Thus, the inventionrelates to such a method which comprises chemically synthesizing one ormore nucleic acid molecules in which recombination sites are added byadding the appropriate sequence of nucleotides during the synthesisprocess.

[0078] In another embodiment of the invention, one or more recombinationsites may be added to nucleic acid molecules of interest by a methodwhich comprises:

[0079] (a) contacting one or more nucleic acid molecules with one ormore integration sequences which comprise one or more recombinationsites or portions thereof; and

[0080] (b) incubation of said mixture under conditions sufficient toincorporate said recombination site containing integration sequencesinto said nucleic acid molecules. In accordance with this aspect of theinvention, integration sequences may comprise any nucleic acid moleculeswhich through recombination or by integration become a part of thenucleic acid molecule of interest. Integration sequences may beintroduced in accordance with this aspect of the invention by in vivo orin vitro recombination (homologous recombination or illegitimaterecombination) or by in vivo or in vitro installation by usingtransposons, insertion sequences, integrating viruses, homing introns,or other integrating elements.

[0081] In another aspect, the invention relates to kits for carrying outthe methods of the invention and more specifically relates to cloning orsubcloning kits and kits for making Insert Donor molecules of theinvention. Such kits may comprise a carrier or receptacle beingcompartmentalized to receive and hold therein any number of containers.Such containers may contain any number of components for carrying outthe methods of the invention or combinations of such components. Inparticular, a kit of the invention may comprise one or more components(or combinations thereof) selected from the group consisting of one ormore recombination proteins or recombinases, one or more Vector Donormolecules, one or more Insert Donor molecules and one or more host cells(e.g. competent cells).

[0082] Kits for making the Insert Donor molecules of the invention maycomprise any or a number of components and the composition of such kitsmay vary depending on the specific method involved. Kits forsynthesizing Insert Donor molecules by amplification may comprise one ormore components (or combinations thereof) selected from the groupconsisting of one or more polypeptides having polymerase activity(preferably DNA polymerases and most preferably thermostable DNApolymerases), one or more nucleotides, and one or more primerscomprising one or more recombination sites. Kits for inserting or addingrecombination sites to nucleic acid molecules of interest may compriseone or more nucleases (preferably restriction endonucleases), one ormore ligases, one or more topoisomerases one or more polymerases, andone or more nucleic acid molecules or adapters comprising one or morerecombination sites. Kits for integrating recombination sites into oneor more nucleic acid molecules of interest may comprise one or morecomponents (or combinations thereof) selected from the group consistingof one or more integration sequences comprising one or morerecombination sites. Such integration sequences may comprise one or moretransposons, integrating viruses, homologous recombination sequences,one or more host cells and the like.

[0083] The invention also relates to compositions for carrying out themethods of the invention or compositions which are produced fromcarrying out the methods of the invention. In particular, suchcompositions may comprise one or more Insert Donor molecules, one ormore Vector Donor molecules and one or more recombination proteins (orcombinations thereof). In a further aspect, the compositions of theinvention may comprise one or more cointegrate molecules, one or moreProduct molecules and one or more Byproduct molecule (or combinationsthereof).

[0084] Compositions related to preparing Insert Donor molecules may varydepending on the particular method utilized in preparing the desiredInsert Donor molecules. Compositions for preparing such molecules byamplification may comprise one or more polypeptides having polymeraseactivity, one or more primers comprising one or more recombinationsites, one or more nucleotides and one or more nucleic acid molecule tobe amplified (or combinations thereof). Compositions related toinserting or adding recombination sites in a desired nucleic acidmolecule may comprise one or more nucleic acid molecules or adapterscomprising one or more recombination sites, one or more ligases, one ormore restriction endonucleases, one or more topoisomerases, and one ormore nucleic acid molecules desired to contain such recombination sites(or coinbinations thereof). Compositions related to integration ofrecombination sites in a desired nucleic acid molecule may comprise oneor more integration sequences comprising one or more recombination sitesand one or more nucleic acid molecules desired to contain therecombination sites.

[0085] In a particularly preferred aspect of the invention, libraries(e.g. populations of genomic DNA or cDNA, or populations of nucleic acidmolecules, produced by de novo synthesis such as random sequences ordegenerate oligonucleotides) are utilized in accordance with the presentinvention. By inserting or adding recombination sites to suchpopulations of nucleic acid molecules, a population of Insert Donormolecules are produced. By the recombination methods of the invention,the library may be easily moved into different vectors (or combinationsof vectors) and thus into different host systems (prokaryotic andeukaryotic) to evaluate and analyze the library or a particularsequences or clones derived from the library. Alternatively, the vectorscontaining the desired molecule may be used in vitro systems such as invitro expression systems for production of RNA and/or protein. In aparticularly preferred aspect, one or more recombination sites are addedto nucleic acid molecules of the library by method comprising:

[0086] (a) mixing a population of linear nucleic acid molecules with oneor more adapters comprising one or more recombination sites; and

[0087] (b) incubating said mixture under conditions sufficient to addone or more of said adapters to one or more termini of said linearmolecules. In a preferred aspect, the population of nucleic acidmolecules are double stranded DNA molecules (preferably genomic DNA orcDNA). A population of linear fragments for use in the invention may beprepared by cleaving (by mechanical or enzymatic means) the genomic orcDNA. In a preferred aspect, the adapters are added to one or moretermini of the linear molecules.

[0088] In another particularly preferred aspect of the invention, cDNAlibraries are used to prepare a population of Insert Donor DNA moleculesof the invention. In particular, this aspect of the invention relates toa method which comprises:

[0089] (a) contacting a population of RNA, mRNA or polyA+ RNA templateswith one or more polypeptides having reverse transcriptase activity andone or more primers which comprises one or more recombination sites;

[0090] (b) incubating said mixture under conditions sufficient tosynthesize a first population of DNA molecules complementary to saidtemplates, wherein said DNA molecules comprise one or more recombinationsites. This aspect of the invention may further comprise incubating saidsynthesized DNA under conditions sufficient to make a second populationof DNA molecules complementary to all or a portion of said firstpopulation of DNA molecules, thereby forming a population of doublestranded DNA molecules comprising one or more recombination sites.

[0091] In a particularly preferred aspect, the Insert Donor molecules ofthe invention comprise at least two recombination sites and where theInsert Donor molecules are linear, such two or more recombination sitesare preferably located at or near both termini of the molecules. Inaccordance with the invention, the use of additional recombination sites(i.e. more than two) may be used to facilitate subcloning of differentinserts within the Insert Donor molecule, depending on the type andplacement of such recombination sites.

[0092] Other embodiments include DNA and vectors useful in the methodsof the present invention. In particular, Vector Donor molecules areprovided in one embodiment, wherein DNA segments within the Vector Donorare separated either by, (i) in a circular Vector Donor, at least tworecombination sites, or (ii) in a linear Vector Donor, at least onerecombination site, where the recombination sites are preferablyengineered to enhance specificity or efficiency of recombination. OneVector Donor embodiment comprises a first DNA segment and a second DNAsegment, the first or second segment comprising a selectable marker. Asecond Vector Donor embodiment comprises a first DNA segment and asecond DNA segment, the first or second DNA segment comprising a toxicgene. A third Vector Donor embodiment comprises a first DNA segment anda second DNA segment, the first or second DNA segment comprising aninactive fragment of at least one selectable marker, wherein theinactive fragment of the Selectable marker is capable of reconstitutinga functional Selectable marker when recombined across the first orsecond recombination site with another inactive fragment of at least oneSelectable marker.

[0093] Other preferred embodiments of the present invention will beapparent to one of ordinary skill in light of what is known in the art,in light of the following drawings and description of the invention, andin light of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0094]FIG. 1 depicts one general method of the present invention,wherein the starting (parent) DNA molecules can be circular or linear.The goal is to exchange the new subcloning vector D for the originalcloning vector B. It is desirable in one embodiment to select for AD andagainst all the other molecules, including the Cointegrate. The squareand circle are sites of recombination: e.g., loxP sites, att sites, etc.For example, segment D can contain expression signals, new drug markers,new origins of replication, or specialized functions for mapping orsequencing DNA.

[0095]FIG. 2A depicts an in vitro method of recombining an Insert Donorplasmid (here, pEZC705) with a Vector Donor plasmid (here, pEZC726), andobtaining Product DNA and Byproduct daughter molecules. The tworecombination sites are attP and loxP on the Vector Donor. On onesegment defined by these sites is a kanamycin resistance gene whosepromoter has been replaced by the tetOP operator/promoter fromtransposon Tn10. See, e.g., Sizemore et al., Nucl. Acids Res.18(10):2875 (1990). In the absence of tet repressor protein, E. coli RNApolymerase transcribes the kanamycin resistance gene from the tetOP. Iftet repressor is present, it binds to tetOP and blocks transcription ofthe kanamycin resistance gene. The other segment of pEZC726 has the tetrepressor gene expressed by a constitutive promoter. Thus cellstransformed by pEZC726 are resistant to chloramphenicol, because of thechloramphenicol acetyl transferase gene on the same segment as tetR, butare sensitive to kanamycin. The recombinase-mediated reactions result inseparation of the tetR gene from the regulated kanamycin resistancegene. This separation results in kanamycin resistance only in cellsreceiving the desired recombination product. The first recombinationreaction is driven by the addition of the recombinase called Integrase.The second recombination reaction is driven by adding the recombinaseCre to the Cointegrate (here, pEZC7 Cointegr).

[0096]FIG. 2B depicts a restriction map of pEZC705.

[0097]FIG. 2C depicts a restriction map of pEZC726.

[0098]FIG. 2D depicts a restriction map of pEZC7 Coint.

[0099]FIG. 2E depicts a restriction map of Intprod.

[0100]FIG. 2F depicts a restriction map of Intbypro.

[0101]FIG. 3A depicts an in vitro method of recombining an Insert Donorplasmid (here, pEZC602) with a Vector Donor plasmid (here, pEZC629), andobtaining Product (here, EZC6prod) and Byproduct (here, EZC6Bypr)daughter molecules. The two recombination sites are loxP and loxP 511.One segment of pEZC629 defined by these sites is a kanamycin resistancegene whose promoter has been replaced by the tetOP operator/promoterfrom transposon Tn10. In the absence of tet repressor protein, E. coliRNA polymerase transcribes the kanamycin resistance gene from the tetOP.If tet repressor is present, it binds to tetOP and blocks transcriptionof the kanamycin resistance gene. The other segment of pEZC629 has thetet repressor gene expressed by a constitutive promoter. Thus cellstransformed by pEZC629 are resistant to chloramphenicol, because of thechloramphenicol acetyl transferase gene on the same segment as tetR, butare sensitive to kanamycin. The reactions result in separation of thetetR gene from the regulated kanamycin resistance gene. This separationresults in kanamycin resistance in cells receiving the desiredrecombination product. The first and the second recombination events aredriven by the addition of the same recombinase, Cre.

[0102]FIG. 3B depicts a restriction map of EZC6Bypr.

[0103]FIG. 3C depicts a restriction map of EZC6prod.

[0104]FIG. 3D depicts a restriction map of pEZC602.

[0105]FIG. 3E depicts a restriction map of pEZC629.

[0106]FIG. 3F depicts a restriction map of EZC6coint.

[0107]FIG. 4A depicts an application of the in vitro method ofrecombinational cloning to subclone the chloramphenicol acetyltransferase gene into a vector for expression in eukaryotic cells. TheInsert Donor plasmid, pEZC843, is comprised of the chloramphenicolacetyl transferase gene of E. coli, cloned between loxP and attB sitessuch that the loxP site is positioned at the 5′-end of the gene. TheVector Donor plasmid, pEZC1003, contains the cytomegalovirus eukaryoticpromoter apposed to a loxP site. The supercoiled plasmids were combinedwith lambda Integrase and Cre recombinase in vitro. After incubation,competent E. coli cells were transformed with the recombinationalreaction solution. Aliquots of transformations were spread on agarplates containing kanamycin to select for the Product molecule (hereCMVProd).

[0108]FIG. 4B depicts a restriction map of pEZC843.

[0109]FIG. 4C depicts a restriction map of pEZC1003.

[0110]FIG. 4D depicts a restriction map of CMVBypro.

[0111]FIG. 4E depicts a restriction map of CMVProd.

[0112]FIG. 4F depicts a restriction map of CMVcoint.

[0113]FIG. 5A depicts a vector diagram of pEZC1301.

[0114]FIG. 5B depicts a vector diagram of pEZC1305.

[0115]FIG. 5C depicts a vector diagram of pEZC1309.

[0116]FIG. 5D depicts a vector diagram of pEZC1313.

[0117]FIG. 5E depicts a vector diagram of pEZC1317.

[0118]FIG. 5F depicts a vector diagram of pEZC1321.

[0119]FIG. 5G depicts a vector diagram of pEZC1405.

[0120]FIG. 5H depicts a vector diagram of pEZC1502.

[0121]FIG. 6A depicts a vector diagram of pEZC1603.

[0122]FIG. 6B depicts a vector diagram of pEZC1706.

[0123]FIG. 7A depicts a vector diagram of pEZC2901.

[0124]FIG. 7B depicts a vector diagram of pEZC2913

[0125]FIG. 7C depicts a vector diagram of pEZC3101.

[0126]FIG. 7D depicts a vector diagram of pEZC1802.

[0127]FIG. 8A depicts a vector diagram of pGEX-2TK.

[0128]FIG. 8B depicts a vector diagram of pEZC3501.

[0129]FIG. 8C depicts a vector diagram of pEZC3601.

[0130]FIG. 8D depicts a vector diagram of pEZC3609.

[0131]FIG. 8E depicts a vector diagram of pEZC3617.

[0132]FIG. 8F depicts a vector diagram of pEZC3606.

[0133]FIG. 8G depicts a vector diagram of pEZC3613.

[0134]FIG. 8H depicts a vector diagram of pEZC3621.

[0135]FIG. 8I depicts a vector diagram of GST-CAT.

[0136]FIG. 8J depicts a vector diagram of GST-phoA.

[0137]FIG. 8K depicts a vector diagram of pEZC3201.

[0138]FIG. 9A depicts a diagram of 5.2 kb PCR prod.

[0139]FIG. 9B depicts a vector diagram of pEZC1202.

[0140]FIG. 9C depicts a vector diagram of 5.2 kb clone.

[0141]FIG. 10A depicts a vector diagram of pEZC5601.

[0142]FIG. 10B depicts a vector diagram of pEZC6701.

[0143]FIG. 10C depicts a vector diagram of attL product.

[0144]FIG. 10D depicts attR product.

[0145]FIG. 11A depicts a vector diagram of pEZC7102.

[0146]FIG. 11B depicts a vector diagram of pEZC7501.

[0147]FIG. 11C depicts the attL product.

[0148]FIG. 12A depicts an amp PCR product with terminal attB sites.

[0149]FIG. 12B depicts a tet PCR product with terminal attB sites.

[0150]FIG. 12C depicts a restriction map of amp7102.

[0151]FIG. 12D depicts a restriction map of tet 7102.

DETAILED DESCRIPTION OF THE INVENTION

[0152] It is unexpectedly discovered in the present invention thatreversible and/or repeatable cloning and subcloning reactions can beused to manipulate nucleic acids to form chimeric nucleic acids usingrecombination proteins and recombination sites. Recombinational cloningaccording to the present invention thus uses recombination proteins withrecombinant nucleic acid molecules having at least one selectedrecombination site for moving or exchanging segments of nucleic acidmolecules, in vitro and in vivo.

[0153] These methods use recombination reactions to generate chimericDNA or RNA molecules that have the desired characteristic(s) and/ornucleic acid segment(s). The methods of the invention provide a means inwhich nucleic acid molecule of interest may be moved or transferred intoany number of vector systems. In accordance with the invention, suchtransfer to various vector systems may be accomplished separately,sequentially or in mass (e.g. into any number of different vectors inone step). The improved specificity, speed and/or yields of the presentinvention facilitates DNA or RNA cloning, subcloning, regulation orexchange useful for any related purpose. Such purposes include in vitrorecombination of DNA or RNA segments and in vitro or in vivo insertionor modification of transcribed, replicated, isolated or genomic DNA orRNA.

[0154] Definitions

[0155] In the description that follows, a number of terms used inrecombinant DNA technology are utilized extensively. In order to providea clear and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

[0156] Byproduct: is a daughter molecule (a new clone produced after thesecond recombination event during the recombinational cloning process)lacking the segment which is desired to be cloned or subcloned.

[0157] Cointegrate: is at least one recombination intermediate nucleicacid molecule of the present invention that contains both parental(starting) molecules. It will usually be circular. In some embodimentsit can be linear.

[0158] Host: is any prokaryotic or eukaryotic organism that can be arecipient of the recombinational cloning Product. A “host,” as the termis used herein, includes prokaryotic or eukaryotic organisms that can begenetically engineered. For examples of such hosts, see Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, New York (1982).

[0159] Insert or Inserts: include the desired nucleic acid segment or apopulation of nucleic acid segments (segment A of FIG. 1) which may bemanipulated by the methods of the present invention. Thus, the termsInsert(s) are meant to include a particular nucleic acid (preferablyDNA) segment or a population of segments. Such Insert(s) can compriseone or more genes.

[0160] Insert Donor: is one of the two parental nucleic acid molecules(e.g. RNA or DNA) of the present invention which carries the Insert. TheInsert Donor molecule comprises the Insert flanked on both sides withrecombination sites. The Insert Donor can be linear or circular. In oneembodiment of the invention, the Insert Donor is a circular DNA moleculeand further comprises a cloning vector sequence outside of therecombination signals (see FIG. 1). When a population of Inserts orpopulation of nucleic acid segments are used to make the Insert Donor, apopulation of Insert Donors result and may be used in accordance withthe invention.

[0161] Product: is one the desired daughter molecules comprising the Aand D sequences which is produced after the second recombination eventduring the recombinational cloning process (see FIG. 1). The Productcontains the nucleic acid which was to be cloned or subcloned. Inaccordance with the invention, when a population of Insert Donors areused, the resulting population of Product molecules will contain all ora portion of the population of Inserts of the Insert Donors andpreferably will contain a representative population of the originalmolecules of the Insert Donors.

[0162] Promoter: is a DNA sequence generally described as the 5′-regionof a gene, located proximal to the start codon. The transcription of anadjacent DNA segment is initiated at the promoter region. A repressiblepromoter's rate of transcription decreases in response to a repressingagent. An inducible promoter's rate of transcription increases inresponse to an inducing agent. A constitutive promoter's rate oftranscription is not specifically regulated, though it can vary underthe influence of general metabolic conditions.

[0163] Recognition sequence: Recognition sequences are particularsequences which a protein, chemical compound, DNA, or RNA molecule(e.g., restriction endonuclease, a modification methylase, or arecombinase) recognizes and binds. In the present invention, arecognition sequence will usually refer to a recombination site. Forexample, the recognition sequence for Cre recombinase is loxP which is a34 base pair sequence comprised of two 13 base pair inverted repeats(serving as the recombinase binding sites) flanking an 8 base pair coresequence. See FIG. 1 of Sauer, B., Current Opinion in Biotechnology5:521-527 (1994). Other examples of recognition sequences are the attB,attP, attL, and attR sequences which are recognized by the recombinaseenzyme λ Integrase. attB is an approximately 25 base pair sequencecontaining two 9 base pair core-type Int binding sites and a 7 base pairoverlap region. attP is an approximately 240 base pair sequencecontaining core-type Int binding sites and arm-type Int binding sites aswell as sites for auxiliary proteins integration host factor (IHF), FISand excisionase (Xis). See Landy, Current Opinion in Biotechnology3:699-707 (1993). Such sites may also be engineered according to thepresent invention to enhance production of products in the methods ofthe invention. When such engineered sites lack the P1 or H1 domains tomake the recombination reactions irreversible (e.g., attR or attP), suchsites may be designated attR′ or attP′ to show that the domains of thesesites have been modified in some way.

[0164] Recombinase: is an enzyme which catalyzes the exchange of DNAsegments at specific recombination sites.

[0165] Recombinational Cloning: is a method described herein, wherebysegments of nucleic acid molecules or populations of such molecules areexchanged, inserted, replaced, substituted or modified, in vitro or invivo.

[0166] Recombination proteins: include excisive or integrative proteins,enzymes, co-factors or associated proteins that are involved inrecombination reactions involving one or more recombination sites. See,Landy (1994), infra.

[0167] Repression cassette: is a nucleic acid segment that contains arepressor of a Selectable marker present in the subcloning vector.

[0168] Selectable marker: is a DNA segment that allows one to select foror against a molecule or a cell that contains it, often under particularconditions. These markers can encode an activity, such as, but notlimited to, production of RNA, peptide, or protein, or can provide abinding site for RNA, peptides, proteins, inorganic and organiccompounds or compositions and the like. Examples of Selectable markersinclude but are not limited to: (1) DNA segments that encode productswhich provide resistance against otherwise toxic compounds (e.g.,antibiotics); (2) DNA segments that encode products which are otherwiselacking in the recipient cell (e.g., tRNA genes, auxotrophic markers);(3) DNA segments that encode products which suppress the activity of agene product; (4) DNA segments that encode products which can be readilyidentified (e.g., phenotypic markers such as β-galactosidase, greenfluorescent protein (GFP), and cell surface proteins); (5) DNA segmentsthat bind products which are otherwise detrimental to cell survivaland/or function; (6) DNA segments that otherwise inhibit the activity ofany of the DNA segments described in Nos. 1-5 above (e.g., antisenseoligonucleotides); (7) DNA segments that bind products that modify asubstrate (e.g. restriction endonucleases); (8) DNA segments that can beused to isolate or identify a desired molecule (e.g. specific proteinbinding sites); (9) DNA segments that encode a specific nucleotidesequence which can be otherwise non-functional (e.g., for PCRamplification of subpopulations of molecules); (10) DNA segments, whichwhen absent, directly or indirectly confer resistance or sensitivity toparticular compounds; and/or (11) DNA segments that encode productswhich are toxic in recipient cells.

[0169] Selection scheme: is any method which allows selection,enrichment, or identification of a desired Product or Product(s) from amixture containing the Insert Donor, Vector Donor, any intermediates(e.g. a Cointegrate), and/or Byproducts. The selection schemes of onepreferred embodiment have at least two components that are either linkedor unlinked during recombinational cloning. One component is aSelectable marker. The other component controls the expression in vitroor in vivo of the Selectable marker, or survival of the cell harboringthe plasmid carrying the Selectable marker. Generally, this controllingelement will be a repressor or inducer of the Selectable marker, butother means for controlling expression of the Selectable marker can beused. Whether a repressor or activator is used will depend on whetherthe marker is for a positive or negative selection, and the exactarrangement of the various DNA segments, as will be readily apparent tothose skilled in the art. A preferred requirement is that the selectionscheme results in selection of or enrichment for only one or moredesired Products. As defined herein, selecting for a DNA moleculeincludes (a) selecting or enriching for the presence of the desired DNAmolecule, and (b) selecting or enriching against the presence of DNAmolecules that are not the desired DNA molecule.

[0170] In one embodiment, the selection schemes (which can be carriedout in reverse) will take one of three forms, which will be discussed interms of FIG. 1. The first, exemplified herein with a Selectable markerand a repressor therefore, selects for molecules having segment D andlacking segment C. The second selects against molecules having segment Cand for molecules having segment D. Possible embodiments of the secondform would have a DNA segment carrying a gene toxic to cells into whichthe in vitro reaction products are to be introduced. A toxic gene can bea DNA that is expressed as a toxic gene product (a toxic protein orRNA), or can be toxic in and of itself. (In the latter case, the toxicgene is understood to carry its classical definition of “heritabletrait”.)

[0171] Examples of such toxic gene products are well known in the art,and include, but are not limited to, restriction endonucleases (e.g.,DpnI), apoptosis-related genes (e.g. ASK1 or members of the bcl-2/ced-9family), retroviral genes including those of the human immunodeficiencyvirus (HIV), defensins such as NP-1, inverted repeats or pairedpalindromic DNA sequences, bacteriophage lytic genes such as those fromφX174 or bacteriophage T4; antibiotic sensitivity genes such as rpsL,antimicrobial sensitivity genes such as pheS, plasmid killer genes,eukaryotic transcriptional vector genes that produce a gene producttoxic to bacteria, such as GATA-1, and genes that kill hosts in theabsence of a suppressing function, e.g., kicB or ccdB. A toxic gene canalternatively be selectable in vitro, e.g., a restriction site.

[0172] Many genes coding for restriction endonucleases operably linkedto inducible promoters are known, and may be used in the presentinvention. See, e.g. U.S. Pat. No. 4,960,707 (DpnI and DpnII); U.S. Pat.Nos. 5,000,333, 5,082,784 and 5,192,675 (KpnI); U.S. Pat. No. 5,147,800(NgoAIII and NgoAI); U.S. Pat. No. 5,179,015 (FspI and HaeIII): U.S.Pat. No. 5,200,333 (HaeII and TaqI); U.S. Pat. No. 5,248,605 (HpaII);U.S. Pat. No. 5,312,746 (ClaI); U.S. Pat. Nos. 5,231,021 and 5,304,480(XhoI and XhoII); U.S. Pat. No. 5,334,526 (AluI); U.S. Pat. No.5,470,740 (NsiI); U.S. Pat. No. 5,534,428 (SstI/SacI); U.S. Pat. No.5,202,248 (NcoI); U.S. Pat. No. 5,139,942 (NdeI); and U.S. Pat. No.5,098,839 (PacI). See also Wilson, G. G., Nucl. Acids Res. 19:2539-2566(1991); and Lunnen, K. D., et al., Gene 74:25-32 (1988).

[0173] In the second form, segment D carries a Selectable marker. Thetoxic gene would eliminate transformants harboring the Vector Donor,Cointegrate, and Byproduct molecules, while the Selectable marker can beused to select for cells containing the Product and against cellsharboring only the Insert Donor.

[0174] The third form selects for cells that have both segments A and Din cis on the same molecule, but not for cells that have both segmentsin trans on different molecules. This could be embodied by a Selectablemarker that is split into two inactive fragments, one each on segments Aand D.

[0175] The fragments are so arranged relative to the recombination sitesthat when the segments are brought together by the recombination event,they reconstitute a functional Selectable marker. For example, therecombinational event can link a promoter with a structural gene, canlink two fragments of a structural gene, or can link genes that encode aheterodimeric gene product needed for survival, or can link portions ofa replicon.

[0176] Site-specific recombinase: is a type of recombinase whichtypically has at least the following four activities (or combinationsthereof): (1) recognition of one or two specific nucleic acid sequences;(2) cleavage of said sequence or sequences; (3) topoisomerase activityinvolved in strand exchange; and (4) ligase activity to reseal thecleaved strands of nucleic acid. See Sauer, B., Current Opinions inBiotechnology 5:521-527 (1994). Conservative site-specific recombinationis distinguished from homologous recombination and transposition by ahigh degree of specificity for both partners. The strand exchangemechanism involves the cleavage and rejoining of specific DNA sequencesin the absence of DNA synthesis (Landy, A. (1989) Ann. Rev. Biochem.58:913-949).

[0177] Subcloning vector: is a cloning vector comprising a circular orlinear nucleic acid molecule which includes preferably an appropriatereplicon. In the present invention, the subcloning vector (segment D inFIG. 1) can also contain functional and/or regulatory elements that aredesired to be incorporated into the final product to act upon or withthe cloned DNA Insert (segment A in FIG. 1). The subcloning vector canalso contain a Selectable marker (preferably DNA).

[0178] Vector: is a nucleic acid molecule (preferably DNA) that providesa useful biological or biochemical property to an Insert. Examplesinclude plasmids, phages, autonomously replicating sequences (ARS),centromeres, and other sequences which are able to replicate or bereplicated in vitro or in a host cell, or to convey a desired nucleicacid segment to a desired location within a host cell. A Vector can haveone or more restriction endonuclease recognition sites at which thesequences can be cut in a determinable fashion without loss of anessential biological function of the vector, and into which a nucleicacid fragment can be spliced in order to bring about its replication andcloning. Vectors can further provide primer sites, e.g., for PCR,transcriptional and/or translational initiation and/or regulation sites,recombinational signals, replicons, Selectable markers, etc. Clearly,methods of inserting a desired nucleic acid fragment which do notrequire the use of homologous recombination, transpositions orrestriction enzymes (such as, but not limited to, UDG cloning of PCRfragments (U.S. Pat. No. 5,334,575, entirely incorporated herein byreference), T:A cloning, and the like) can also be applied to clone afragment into a cloning vector to be used according to the presentinvention. The cloning vector can further contain one or more selectablemarkers suitable for use in the identification of cells transformed withthe cloning vector.

[0179] Vector Donor: is one of the two parental nucleic acid molecules(e.g. RNA or DNA) of the present invention which carries the DNAsegments comprising the DNA vector which is to become part of thedesired Product. The Vector Donor comprises a subcloning vector D (or itcan be called the cloning vector if the Insert Donor does not alreadycontain a cloning vector) and a segment C flanked by recombination sites(see FIG. 1). Segments C and/or D can contain elements that contributeto selection for the desired Product daughter molecule, as describedabove for selection schemes. The recombination signals can be the sameor different, and can be acted upon by the same or differentrecombinases. In addition, the Vector Donor can be linear or circular.

[0180] Primer: refers to a single stranded or double strandedoligonucleotide that is extended by covalent bonding of nucleotidemonomers during amplification or polymerization of a nucleic acidmolecule (e.g. a DNA molecule). In a preferred aspect, the primercomprises one or more recombination sites or portions of suchrecombination sites. Portions of recombination sties comprise at least 2bases, at least 5 bases, at least 10 bases or at least 20 bases of therecombination sites of interest. When using portions of recombinationsites, the missing portion of the recombination site may be provided bythe newly synthesized nucleic acid molecule. Such recombination sitesmay be located within and/or at one or both termini of the primer.Preferably, additional sequences are added to the primer adjacent to therecombination site(s) to enhance or improve recombination and/or tostabilize the recombination site during recombination. Suchstabilization sequences may be any sequences (preferably G/C richsequences) of any length. Preferably, such sequences range in size from1 to about 1000 bases, 1 to about 500 bases, and 1 to about 100 bases, 1to about 60 bases, 1 to about 25, 1 to about 10, 2 to about 10 andpreferably about 4 bases. Preferably, such sequences are greater than 1base in length and preferably greater than 2 bases in length.

[0181] Template: refers to double stranded or single stranded nucleicacid molecules which are to be amplified, synthesized or sequenced. Inthe case of double stranded molecules, denaturation of its strands toform a first and a second strand is preferably performed before thesemolecules will be amplified, synthesized or sequenced, or the doublestranded molecule may be used directly as a template. For singlestranded templates, a primer complementary to a portion of the templateis hybridized under appropriate conditions and one or more polypeptideshaving polymerase activity (e.g. DNA polymerases and/or reversetranscriptases) may then synthesize a nucleic acid moleculecomplementary to all or a portion of said template. Alternatively, fordouble stranded templates, one or more promoters may be used incombination with one or more polymerases to make nucleic acid moleculescomplementary to all or a portion of the template. The newly synthesizedmolecules, according to the invention, may be equal or shorter in lengththan the original template. Additionally, a population of nucleic acidtemplates may be used during synthesis or amplification to produce apopulation of nucleic acid molecules typically representative of theoriginal template population.

[0182] Adapter: is an oligonucleotide or nucleic acid fragment orsegment (preferably DNA) which comprises one or more recombination sites(or portions of such recombination sites) which in accordance with theinvention can be added to a circular or linear Insert Donor molecule aswell as other nucleic acid molecules described herein. When usingportions of recombination sites, the missing portion may be provided bythe Insert Donor molecule. Such adapters may be added at any locationwithin a circular or linear molecule, although the adapters arepreferably added at or near one or both termini of a linear molecule.Preferably, adapters are positioned to be located on both sides(flanking) a particularly nucleic acid molecule of interest. Inaccordance with the invention, adapters may be added to nucleic acidmolecules of interest by standard recombinant techniques (e.g.restriction digest and ligation). For example, adapters may be added toa circular molecule by first digesting the molecule with an appropriaterestriction enzyme, adding the adapter at the cleavage site andreforming the circular molecule which contains the adapter(s) at thesite of cleavage. Alternatively, adapters may be ligated directly to oneor more and preferably both termini of a linear molecule therebyresulting in linear molecule(s) having adapters at one or both termini.In one aspect of the invention, adapters may be added to a population oflinear molecules, (e.g. a cDNA library or genomic DNA which has beencleaved or digested) to form a population of linear molecules containingadapters at one and preferably both termini of all or substantialportion of said population.

[0183] Library: refers to a collection of nucleic acid molecules(circular or linear). In one preferred embodiment, a library isrepresentative of all or a significant portion of the DNA content of anorganism (a “genomic” library), or a set of nucleic acid moleculesrepresentative of all or a significant portion of the expressed genes (acDNA library) in a cell, tissue, organ or organism. A library may alsocomprise random sequences made by de novo synthesis, mutagenesis of oneor more sequences and the like. Such libraries may or may not becontained in one or more vectors.

[0184] Amplification: refers to any in vitro method for increasing anumber of copies of a nucleotide sequence with the use of a polymerase.Nucleic acid amplification results in the incorporation of nucleotidesinto a DNA and/or RNA molecule or primer thereby forming a new moleculecomplementary to a template. The formed nucleic acid molecule and itstemplate can be used as templates to synthesize additional nucleic acidmolecules. As used herein, one amplification reaction may consist ofmany rounds of replication. DNA amplification reactions include, forexample, polymerase chain reaction (PCR). One PCR reaction may consistof 5-100 “cycles” of denaturation and synthesis of a DNA molecule.

[0185] Oligonucleotide: refers to a synthetic or natural moleculecomprising a covalently linked sequence of nucleotides which are joinedby a phosphodiester bond between the 3′ position of the deoxyribose orribose of one nucleotide and the 5′ position of the deoxyribose orribose of the adjacent nucleotide.

[0186] Nucleotide: refers to a base-sugar-phosphate combination.Nucleotides are monomeric units of a nucleic acid sequence (DNA andRNA). The term nucleotide includes ribonucleoside triphosphatase ATP,UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP,dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivativesinclude, for example, [αS]dATP, 7-deaza-dGTP and 7-deaza-dATP. The termnucleotide as used herein also refers to dideoxyribonucleosidetriphosphates (ddNTPs) and their derivatives. Illustrated examples ofdideoxyribonucleoside triphosphates include, but are not limited to,ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According to the presentinvention, a “nucleotide” may be unlabeled or detectably labeled by wellknown techniques. Detectable labels include, for example, radioactiveisotopes, fluorescent labels, chemiluminescent labels, bioluminescentlabels and enzyme labels.

[0187] Hybridization: The terms “hybridization” and “hybridizing” refersto base pairing of two complementary single-stranded nucleic acidmolecules (RNA and/or DNA) to give a double stranded molecule. As usedherein, two nucleic acid molecules may be hybridized, although the basepairing is not completely complementary. Accordingly, mismatched basesdo not prevent hybridization of two nucleic acid molecules provided thatappropriate conditions, well known in the art, are used.

[0188] Other terms used in the fields of recombinant DNA technology andmolecular and cell biology as used herein will be generally understoodby one of ordinary skill in the applicable arts.

[0189] Recombination Schemes

[0190] One general scheme for an in vitro or in vivo method of theinvention is shown in FIG. 1, where the Insert Donor and the VectorDonor can be either circular or linear DNA, but is shown as circular.Vector D is exchanged for the original cloning vector B. The InsertDonor need not comprise a vector. The method of the invention allows theInserts A to be transferred into any number of vectors. According to theinvention, the Inserts may be transferred to a particular Vector or maybe transferred to a number of vectors in one step. Additionally, theInserts may be transferred to any number of vectors sequentially, forexample, by using the Product DNA molecule as the Insert Donor incombination with a different Vector Donor. The nucleic acid molecule ofinterest may be transferred into a new vector thereby producing a newProduct DNA molecule. The new Product DNA molecule may then be used asstarting material to transfer the nucleic acid molecule of interest intoa new vector. Such sequential transfers can be performed a number oftimes in any number of different vectors. Thus the invention allows forcloning or subcloning nucleic acid molecules and because of the ease andsimplicity, these methods are particularly suited for high through-putapplications. In accordance with the invention, it is desirable toselect for the daughter molecule containing elements A and D and againstother molecules, including one or more Cointegrate(s). The square andcircle are different sets of recombination sites (e.g., lox sites or attsites). Segment A or D can contain at least one Selection Marker,expression signals, origins of replication, or specialized functions fordetecting, selecting, expressing, mapping or sequencing DNA, where D isused in this example. This scheme can also be reversed according to thepresent invention, as described herein. The resulting product of thereverse reaction (e.g. the Insert Donor) may then be used in combinationwith one or a number of vectors to produce new product molecules inwhich the Inserts are contained by any number of vectors.

[0191] Examples of desired DNA segments that can be part of Element A orD include, but are not limited to, PCR products, large DNA segments,genomic clones or fragments, cDNA clones or fragments, functionalelements, etc., and genes or partial genes, which encode useful nucleicacids or proteins. Moreover, the recombinational cloning of the presentinvention can be used to make ex vivo and in vivo gene transfer vehiclesfor protein expression (native or fusion proteins) and/or gene therapy.

[0192] In FIG. 1, the scheme provides the desired Product as containingA and Vector D, as follows. The Insert Donor (containing A and B) isfirst recombined at the square recombination sites by recombinationproteins, with the Vector Donor (containing C and D), to form aCo-integrate having each of A-D-C-B. Next, recombination occurs at thecircle recombination sites to form Product DNA (A and D) and ByproductDNA (C and B). However, if desired, two or more different Co-integratescan be formed to generate two or more Products.

[0193] In one embodiment of the present in vitro or in vivorecombinational cloning method, a method for selecting at least onedesired Product DNA is provided. This can be understood by considerationof the map of plasmid pEZC726 depicted in FIG. 2. The two exemplaryrecombination sites are attP and loxP. On one segment defined by thesesites is a kanamycin resistance gene whose promoter has been replaced bythe tetOP operator/promoter from transposon Tn10. In the absence of tetrepressor protein, E. coli RNA polymerase transcribes the kanamycinresistance gene from the tetOP. If tet repressor is present, it binds totetOP and blocks transcription of the kanamycin resistance gene. Theother segment of pEZC726 has the tet repressor gene expressed by aconstitutive promoter. Thus cells transformed by pEZC726 are resistantto chloramphenicol, because of the chloramphenicol acetyl transferasegene on the same segment as tetR, but are sensitive to kanamycin. Therecombination reactions result in separation of the tetR gene from theregulated kanamycin resistance gene. This separation results inkanamycin resistance in cells receiving the desired recombinationProduct.

[0194] Two different sets of plasmids were constructed to demonstratethe in vitro method. One set, for use with Cre recombinase only (cloningvector 602 and subcloning vector 629 (FIG. 3)) contained loxP and loxP511 sites. A second set, for use with Cre and integrase (cloning vector705 and subcloning vector 726 (FIG. 2)) contained loxP and att sites.The efficiency of production of the desired daughter plasmid was about60 fold higher using both enzymes than using Cre alone. Nineteen oftwenty four colonies from the Cre-only reaction contained the desiredproduct, while thirty eight of thirty eight colonies from the integraseplus Cre reaction contained the desired product plasmid.

[0195] A variety of other selection schemes can be used that are knownin the art as they can suit a particular purpose for which therecombinational cloning is carried out. Depending upon individualpreferences and needs, a number of different types of selection schemescan be used in the recombinational cloning or subcloning methods of thepresent invention. The skilled artisan can take advantage of theavailability of the many DNA segments or methods for making them and thedifferent methods of selection that are routinely used in the art. SuchDNA segments include but are not limited to those which encodes anactivity such as, but not limited to, production of RNA, peptide, orprotein, or providing a binding site for such RNA, peptide, or protein.Examples of DNA molecules used in devising a selection scheme are givenabove, under the definition of “selection scheme”

[0196] Additional examples include but are not limited to:

[0197] (i) Generation of new primer sites for PCR (e.g., juxtapositionof two DNA sequences that were not previously juxtaposed);

[0198] (ii) Inclusion of a DNA sequence acted upon by a restrictionendonuclease or other DNA modifying enzyme, chemical, ribozyme, etc.;

[0199] (iii) Inclusion of a DNA sequence recognized by a DNA bindingprotein, RNA, DNA, chemical, etc.) (e.g., for use as an affinity tag forselecting for or excluding from a population (Davis, Nucl. Acids Res.24:702-706 (1996); J. Virol. 69: 8027-8034 (1995)) or for juxtaposing apromoter for in vitro transcription;

[0200] (iv) In vitro selection of RNA ligands for the ribosomal L22protein associated with Epstein-Barr virus-expressed RNA by usingrandomized and cDNA-derived RNA libraries;

[0201] (vi) The positioning of functional elements whose activityrequires a specific orientation or juxtaposition (e.g., (a) arecombination site which reacts poorly in trans, but when placed in cis,in the presence of the appropriate proteins, results in recombinationthat destroys certain populations of molecules; (e.g., reconstitution ofa promoter sequence that allows in vitro RNA synthesis). The RNA can beused directly, or can be reverse transcribed to obtain the desired DNAconstruct;

[0202] (vii) Selection of the desired product by size (e.g.,fractionation) or other physical property of the molecule(s); and

[0203] (viii) Inclusion of a DNA sequence required for a specificmodification (e.g., methylation) that allows its identification.

[0204] After formation of the Product and Byproduct in the method of thepresent invention, the selection step can be carried out either in vitroor in vivo depending upon the particular selection scheme which has beenoptionally devised in the particular recombinational cloning procedure.

[0205] For example, an in vitro method of selection can be devised forthe Insert Donor and Vector Donor DNA molecules. Such scheme can involveengineering a rare restriction site in the starting circular vectors insuch a way that after the recombination events the rare cutting sitesend up in the Byproduct. Hence, when the restriction enzyme which bindsand cuts at the rare restriction site is added to the reaction mixturein vitro, all of the DNA molecules carrying the rare cutting site, i.e.,the starting DNA molecules, the Cointegrate, and the Byproduct, will becut and rendered nonreplicable in the intended host cell. For example,cutting sites in segments B and C (see FIG. 1) can be used to selectagainst all molecules except the Product. Alternatively, only a cuttingsite in C is needed if one is able to select for segment D, e.g., by adrug resistance gene not found on B.

[0206] Similarly, an in vitro selection method can be devised whendealing with linear DNA molecules. DNA sequences complementary to a PCRprimer sequence can be so engineered that they are transferred, throughthe recombinational cloning method, only to the Product molecule. Afterthe reactions are completed, the appropriate primers are added to thereaction solution and the sample is subjected to PCR. Hence, all or partof the Product molecule is amplified.

[0207] Other in vivo selection schemes can be used with a variety ofhost cells, particularly E. coli lines. One is to put a repressor geneon one segment of the subcloning plasmid, and a drug marker controlledby that repressor on the other segment of the same plasmid. Another isto put a killer gene on segment C of the subcloning plasmid (FIG. 1). Ofcourse a way must exist for growing such a plasmid, i.e., there mustexist circumstances under which the killer gene will not kill. There area number of these genes known which require particular strains of E.coli. One such scheme is to use the restriction enzyme DpnI, which willnot cleave unless its recognition sequence GATC is methylated. Manypopular common E. coli strains methylate GATC sequences, but there aremutants in which cloned DpnI can be expressed without harm. Otherrestriction enzyme genes may also be used as a toxic gene for selection.In such cases, a host containing a gem encoding the correspondingmethylase gene provides protected host for use in the invention.Similarly, the ccdB protein is a potent poison of DNA gyrase,efficiently trapping gyrase molecules in a cleavable complex, resultingin DNA strand breakage and cell death. Mutations in the gyrA subunit ofDNA gyrase, specifically the gyrA462 mutation, confers resistance toccdB (Bernard and Couturier, J. Mol. Bio. 226 (1992) 735-745). An E.coli strain, DB2, has been constructed that contains the gyrA462mutation. DB2 cells containing plasmids that express the ccdB gene arenot killed by ccd B. This strain is available from Life Technologies andhas been deposited on Oct. 14, 1997 with the Collection, AgriculturalResearch Culture Collection (NRRL), 1815 North University Street,Peoria, Ill. 61604 USA as deposit number NRRL B-21852.

[0208] Of course analogous selection schemes can be devised for otherhost organisms. For example, the tet repressor/operator of Tn10 has beenadapted to control gene expression in eukaryotes (Gossen, M., andBujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)). Thus thesame control of drug resistance by the tet repressor exemplified hereinor other selection schemes described herein can be applied to select forProduct in eukaryotic cells.

[0209] Recombination Proteins

[0210] In the present invention, the exchange of DNA segments isachieved by the use of recombination proteins, including recombinasesand associated co-factors and proteins. Various recombination proteinsare described in the art. Examples of such recombinases include:

[0211] Cre: A protein from bacteriophage P1 (Abremski and Hoess, J.Biol. Chem. 259(3):1509-1514 (1984)) catalyzes the exchange (i.e.,causes recombination) between 34 bp DNA sequences called loxP (locus ofcrossover) sites (See Hoess et al., Nucl. Acids Res. 14(5):2287 (1986)).Cre is available commercially (Novagen, Catalog No. 69247-1).Recombination mediated by Cre is freely reversible. From thermodynamicconsiderations it is not surprising that Cre-mediated integration(recombination between two molecules to form one molecule) is much lessefficient than Cre-mediated excision (recombination between two loxPsites in the same molecule to form two daughter molecules). Cre works insimple buffers with either magnesium or spermidine as a cofactor, as iswell known in the art. The DNA substrates can be either linear orsupercoiled. A number of mutant loxP sites have been described (Hoess etal., supra). One of these, loxP 511, recombines with another loxP 511site, but will not recombine with a loxP site.

[0212] Integrase: A protein from bacteriophage lambda that mediates theintegration of the lambda genome into the E. coli chromosome. Thebacteriophage λ Int recombinational proteins promote recombinationbetween its substrate att sites as part of the formation or induction ofa lysogenic state. Reversibility of the recombination reactions resultsfrom two independent pathways for integrative and excisiverecombination. Each pathway uses a unique, but overlapping, set of the15 protein binding sites that comprise att site DNAs. Cooperative andcompetitive interactions involving four proteins (Int, Xis, IHF and FIS)determine the direction of recombination.

[0213] Integrative recombination involves the Int and IHF proteins andsites attP (240 bp) and attB (25 bp). Recombination results in theformation of two new sites: attL and attR. Excisive recombinationrequires Int, IHF, and Xis, and sites attL and attR to generate attP andattB. Under certain conditions, FIS stimulates excisive recombination.In addition to these normal reactions, it should be appreciated thatattP and attB, when placed on the same molecule, can promote excisiverecombination to generate two excision products, one with attL and onewith attR. Similarly, intermolecular recombination between moleculescontaining attL and attR, in the presence of Int, IHF and Xis, canresult in integrative recombination and the generation of attP and attB.Hence, by flanking DNA segments with appropriate combinations ofengineered att sites, in the presence of the appropriate recombinationproteins, one can direct excisive or integrative recombination, asreverse reactions of each other.

[0214] Each of the att sites contains a 15 bp core sequence; individualsequence elements of functional significance lie within, outside, andacross the boundaries of this common core (Landy, A., Ann. Rev. Biochem.58:913 (1989)). Efficient recombination between the various att sitesrequires that the sequence of the central common region be identicalbetween the recombining partners, however, the exact sequence is nowfound to be modifiable. Consequently, derivatives of the att site withchanges within the core are now discovered to recombine as least asefficiently as the native core sequences.

[0215] Integrase acts to recombine the attP site on bacteriophage lambda(about 240 bp) with the attB site on the E. coli genome (about 25 bp)(Weisberg, R. A. and Landy, A. in Lambda II, p. 211 (1983), Cold SpringHarbor Laboratory)), to produce the integrated lambda genome flanked byattL (about 100 bp) and attR (about 160 bp) sites. In the absence of Xis(see below), this reaction is essentially irreversible. The integrationreaction mediated by integrase and IHF works in vitro, with simplebuffer containing spermidine. Integrase can be obtained as described byNash, H. A., Methods of Enzymology 100:210-216 (1983). IHF can beobtained as described by Filutowicz, M., et al., Gene 147:149-150(1994).

[0216] Numerous recombination systems from various organisms can also beused, based on the teaching and guidance provided herein. See, e.g.,Hoess et al., Nucleic Acids Research 14(6):2287 (1986); Abremski et al.,J. Biol. Chem.261(1):391 (1986); Campbell, J. Bacteriol. 174(23):7495(1992); Qian et al., J. Biol. Chem. 267(11):7794 (1992); Araki et al.,J. Mol. Biol. 225(1):25 (1992)). Many of these belong to the integrasefamily of recombinases (Argos et al. EMBO J. 5:433-440 (1986)). Perhapsthe best studied of these are the Integrase/att system frombacteriophage λ (Landy, A. (1993) Current Opinions in Genetics andDevel. 3:699-707), the Cre/loxP system from bacteriophage P1 (Hoess andAbremski (1990) In Nucleic Acids and Molecular Biology, vol. 4. Eds.:Eckstein and Lilley, Berlin-Heidelberg: Springer-Verlag; pp. 90-109),and the FLP/FRT system from the Saccharomyces cerevisiae 2μ circleplasmid (Broach et al. Cell 29:227-234 (1982)).

[0217] Members of a second family of site-specific recombinases, theresolvase family (e.g., γδ, Tn3 resolvase, Hin, Gin, and Cin) are alsoknown. Members of this highly related family of recombinases aretypically constrained to intramolecular reactions (e.g., inversions andexcisions) and can require host-encoded factors. Mutants have beenisolated that relieve some of the requirements for host factors (Maeserand Kahnmann (1991) Mol. Gen. Genet. 230:170-176), as well as some ofthe constraints of intramolecular recombination.

[0218] Other site-specific recombinases similar to λ Int and similar toP1 Cre can be substituted for Int and Cre. Such recombinases are known.In many cases the purification of such other recombinases has beendescribed in the art. In cases when they are not known, cell extractscan be used or the enzymes can be partially purified using proceduresdescribed for Cre and Int.

[0219] While Cre and Int are described in detail for reasons of example,many related recombinase systems exist and their application to thedescribed invention is also provided according to the present invention.The integrase family of site-specific recombinases can be used toprovide alternative recombination proteins and recombination sites forthe present invention, as site-specific recombination proteins encodedby, for example bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1.This group of proteins exhibits an unexpectedly large diversity ofsequences. Despite this diversity, all of the recombinases can bealigned in their C-terminal halves. A 40-residue region near the Cterminus is particularly well conserved in all the proteins and ishomologous to a region near the C terminus of the yeast 2 mu plasmid Flpprotein. Three positions are perfectly conserved within this family:histidine, arginine and tyrosine are found at respective alignmentpositions 396, 399 and 433 within the well-conserved C-terminal region.These residues contribute to the active site of this family ofrecombinases, and suggest that tyrosine-433 forms a transient covalentlinkage to DNA during strand cleavage and rejoining. See, e.g., Argos,P. et al., EMBO J. 5:433-40 (1986).

[0220] The recombinases of some transposons, such as those ofconjugative transposons (e.g., Tn916) (Scott and Churchward. 1995. AnnRev Microbiol 49:367; Taylor and Churchward, 1997. J Bacteriol 179:1837)belong to the integrase family of recombinases and in some cases showstrong preferences for specific integration sites (Ike et al 1992. JBacteriol 174:1801; Trieu-Cuot et al, 1993. Mol. Microbiol 8:179).

[0221] Alternatively, IS231 and other Bacillus thuringiensistransposable elements could be used as recombination proteins andrecombination sites. Bacillus thuringiensis is an entomopathogenicbacterium whose toxicity is due to the presence in the sporangia ofdelta-endotoxin crystals active against agricultural pests and vectorsof human and animal diseases. Most of the genes coding for these toxinproteins are plasmid-borne and are generally structurally associatedwith insertion sequences (IS231, IS232, IS240, ISBT1 and ISBT2) andtransposons (Tn4430 and Tn5401). Several of these mobile elements havebeen shown to be active and participate in the crystal gene mobility,thereby contributing to the variation of bacterial toxicity.

[0222] Structural analysis of the iso-IS231 elements indicates that theyare related to IS1151 from Clostridium perfringens and distantly relatedto IS4 and IS186 from Escherichia coli. Like the other IS4 familymembers, they contain a conserved transposase-integrase motif found inother IS families and retroviruses. Moreover, functional data gatheredfrom IS231A in Escherichia coli indicate a non-replicative mode oftransposition, with a preference for specific targets. Similar resultswere also obtained in Bacillus subtilis and B. thuringiensis. See, e.g.,Mahillon, J. et al., Genetica 93:13-26 (1994); Campbell, J. Bacteriol.7495-7499 (1992).

[0223] An unrelated family of recombinases, the transposases, have alsobeen used to transfer genetic information between replicons. Transposonsare structurally variable, being described as simple or compound, buttypically encode the recombinase gene flanked by DNA sequences organizedin inverted orientations. Integration of transposons can be random orhighly specific. Representatives such as Tn7, which are highlysite-specific, have been applied to the efficient movement of DNAsegments between replicons (Lucklow et al. 1993. J. Virol 67:4566-4579).

[0224] A related element, the integron, are alsotranslocatable-promoting movement of drug resistance cassettes from onereplicon to another. Often these elements are defective transposonderivatives. Transposon Tn21 contains a class I integron called In2. Theintegrase (IntI1) from In2 is common to all integrons in this class andmediates recombination between two 59-bp elements or between a 59-bpelement and an attI site that can lead to insertion into a recipientintegron. The integrase also catalyzes excisive recombination. (Hall,1997. Ciba Found Symp 207:192; Francia et al., 1997. J Bacteriol179:4419).

[0225] Group II introns are mobile genetic elements encoding a catalyticRNA and protein. The protein component possesses reverse transcriptase,maturase and an endonuclease activity, while the RNA possessesendonuclease activity and determines the sequence of the target siteinto which the intron integrates. By modifying portions of the RNAsequence, the integration sites into which the element integrates can bedefined. Foreign DNA sequences can be incorporated between the ends ofthe intron, allowing targeting to specific sites. This process, termedretrohoming, occurs via a DNA:RNA intermediate, which is copied intocDNA and ultimately into double stranded DNA (Matsuura et al., Genes andDev 1997; Guo et al, EMBO J, 1997). Numerous intron-encoded homingendonucleases have been identified (Belfort and Roberts, 1997. NAR25:3379).Such systems can be easily adopted for application to thedescribed subcloning methods.

[0226] The amount of recombinase which is added to drive therecombination reaction can be determined by using known assays.Specifically, titration assay is used to determine the appropriateamount of a purified recombinase enzyme, or the appropriate amount of anextract.

[0227] Engineered Recombination Sites

[0228] The above recombinases and corresponding recombinase sites aresuitable for use in recombination cloning according to the presentinvention. However, wild-type recombination sites may contain sequencesthat reduce the efficiency or specificity of recombination reactions orthe function of the Product molecules as applied in methods of thepresent invention. For example, multiple stop codons in attB, attR,attP, attL and loxP recombination sites occur in multiple reading frameson both strands, so translation efficiencies are reduced, e.g., wherethe coding sequence must cross the recombination sites, (only onereading frame is available on each strand of loxP and attB sites) orimpossible (in attP, attR or attL).

[0229] Accordingly, the present invention also provides engineeredrecombination sites that overcome these problems. For example, att sitescan be engineered to have one or multiple mutations to enhancespecificity or efficiency of the recombination reaction and theproperties of Product DNAs (e.g., att1, att2, and att3 sites); todecrease reverse reaction (e.g., removing P1 and H1 from attR). Thetesting of these mutants determines which mutants yield sufficientrecombinational activity to be suitable for recombination subcloningaccording to the present invention.

[0230] Mutations can therefore be introduced into recombination sitesfor enhancing site specific recombination. Such mutations include, butare not limited to: recombination sites without translation stop codonsthat allow fusion proteins to be encoded; recombination sites recognizedby the same proteins but differing in base sequence such that they reactlargely or exclusively with their homologous partners allowing multiplereactions to be contemplated; and mutations that prevent hairpinformation of recombination sites. Which particular reactions take placecan be specified by which particular partners are present in thereaction mixture. For example, a tripartite protein fusion could beaccomplished with parental plasmids containing recombination sites attR1and attL1; and attB3; attR1; attP3 and 10×P; and/or attR3 and 10×P;and/or attR3 and attL2.

[0231] There are well known procedures for introducing specificmutations into nucleic acid sequences. A number of these are describedin Ausubel, F. M. et al., Current Protocols in Molecular Biology, WileyInterscience, New York (1989-1996). Mutations can be designed intooligonucleotides, which can be used to modify existing cloned sequences,or in amplification reactions. Random mutagenesis can also be employedif appropriate selection methods are available to isolate the desiredmutant DNA or RNA. The presence of the desired mutations can beconfirmed by sequencing the nucleic acid by well known methods.

[0232] The following non-limiting methods can be used to modify ormutate a core region of a given recombination site to provide mutatedsites that can be used in the present invention:

[0233] 1. By recombination of two parental DNA sequences bysite-specific (e.g. attL and attR to give attB) or other (e.g.homologous) recombination mechanisms where the parental DNA segmentscontain one or more base alterations resulting in the final mutated coresequence;

[0234] 2. By mutation or mutagenesis (site-specific, PCR, random,spontaneous, etc) directly of the desired core sequence;

[0235] 3. By mutagenesis (site-specific, PCR, random, spontaneous, etc)of parental DNA sequences, which are recombined to generate a desiredcore sequence;

[0236] 4. By reverse transcription of an RNA encoding the desired coresequence; and

[0237] 5. By de novo synthesis (chemical synthesis) of a sequence havingthe desired base changes.

[0238] The functionality of the mutant recombination sites can bedemonstrated in ways that depend on the particular characteristic thatis desired. For example, the lack of translation stop codons in arecombination site can be demonstrated by expressing the appropriatefusion proteins. Specificity of recombination between homologouspartners can be demonstrated by introducing the appropriate moleculesinto in vitro reactions, and assaying for recombination products asdescribed herein or known in the art. Other desired mutations inrecombination sites might include the presence or absence of restrictionsites, translation or transcription start signals, protein bindingsites, and other known functionalities of nucleic acid base sequences.Genetic selection schemes for particular functional attributes in therecombination sites can be used according to known method steps. Forexample, the modification of sites to provide (from a pair of sites thatdo not interact) partners that do interact could be achieved byrequiring deletion, via recombination between the sites, of a DNAsequence encoding a toxic substance. Similarly, selection for sites thatremove translation stop sequences, the presence or absence of proteinbinding sites, etc., can be easily devised by those skilled in the art.

[0239] Accordingly, the present invention provides a nucleic acidmolecule, comprising at least one DNA segment having at least twoengineered recombination sites flanking a Selectable marker and/or adesired DNA segment, wherein at least one of said recombination sitescomprises a core region having at least one engineered mutation thatenhances recombination in vitro in the formation of a Cointegrate DNA ora Product DNA.

[0240] While in the preferred embodiment the recombination sites differin sequence and do not interact with each other, it is recognized thatsites comprising the same sequence can be manipulated to inhibitrecombination with each other. Such conceptions are considered andincorporated herein. For example, a protein binding site can beengineered adjacent to one of the sites. In the presence of the proteinthat recognizes said site, the recombinase fails to access the site andthe other site is therefore used preferentially. In the cointegrate thissite can no longer react since it has been changed e.g. from attB toattL. In resolution of the cointegrate, the protein can be inactivated(e.g. by antibody, heat or a change of buffer) and the second site canundergo recombination.

[0241] The nucleic acid molecule can have at least one mutation thatconfers at least one enhancement of said recombination, said enhancementselected from the group consisting of substantially (i) favoringintegration; (ii) favoring recombination; (ii) relieving the requirementfor host factors; (iii) increasing the efficiency of said CointegrateDNA or Product DNA formation; and (iv) increasing the specificity ofsaid Cointegrate DNA or Product DNA formation.

[0242] The nucleic acid molecule preferably comprises at least onerecombination site derived from attB, attP, attL or attR, such as attR′or attP′. More preferably the att site is selected from att1, att2, oratt3, as described herein.

[0243] In a preferred embodiment, the core region comprises a DNAsequence selected from the group consisting of: (a)RKYCWGCTTTYKTRTACNAASTSGB (SEQ ID NO: 1) (m-att); (b)AGCCWGCTTTYKTRTACNAACTSGB (SEQ ID NO: 2) (m-attB); (c)GTTCAGCTTTCKTRTACNAACTSGB (SEQ ID NO: 3) (m-attR); (d)AGCCWGCTTTCKTRTACNAAGTSGB (SEQ ID NO: 4) (m-attL); (e)GTTCAGCTTTYKTRTACNAAGTSGB (SEQ ID NO: 5) (m-attP1); (f) RBYCWGCTTTYTTRTACWAA STKGD (SEQ ID NO: 39) (n-att); (g) ASCCW GCTTTYTTRTACWAASTKGW (SEQ ID NO: 40) (n-attB); (h) ASCCW GCTTTYTTRTACWAA GTTGG (SEQ IDNO: 41) (n-attL); (i) GTTCA GCTTTYTTRTACWAA STKGW (SEQ ID NO: 42)(n-attR); (j) GTTCA GCTTTYTTRTACWAA GTTGG (SEQ ID NO: 43) (n-attP);

[0244] or a corresponding or complementary DNA or RNA sequence, whereinR=A or G; K=G or T/U; Y═C or T/U; W=A or T/U; N=A or C or G or T/U; S═Cor G; and B═C or G or T/U, as presented in 37 C.F.R. §1.822, which isentirely incorporated herein by reference, wherein the core region doesnot contain a stop codon in one or more reading frames.

[0245] The core region also preferably comprises a DNA sequence selectedfrom the group consisting of: (a) AGCCTGCTTTTTTGTACAAACTTGT (SEQ ID NO:6) (attB1); (b) AGCCTGCTTTCTTGTACAAACTTGT (SEQ ID NO: 7) (attB2); (c)ACCCAGCTTTCTTGTACAAAGTGGT (SEQ ID NO: 8) (attB3); (d)GTTCAGCTTTTTTGTACAAACTTGT (SEQ ID NO: 9) (attR1); (e)GTTCAGCTTTCTTGTACAAACTTGT (SEQ ID NO: 10) (attR2); (f)GTTCAGCTTTCTTGTACAAAGTGGT (SEQ ID NO: 11) (attR3); (g)AGCCTGCTTTTTTGTACAAAGTTGG (SEQ ID NO: 12) (attL1); (h)AGCCTGCTTTCTTGTACAAAGTTGG (SEQ ID NO: 13) (attL2); (i)ACCCAGCTTTCTTGTACAAAGTTGG (SEQ ID NO: 14) (attL3); (j)GTTCAGCTTTTTTGTACAAAGTTGG (SEQ ID NO: 15) (attP1); (k)GTTCAGCTTTCTTGTACAAAGTTGG (SEQ ID NO: 16) (attP2, P3);

[0246] or a corresponding or complementary DNA or RNA sequence.

[0247] The present invention thus also provides a method for making anucleic acid molecule, comprising providing a nucleic acid moleculehaving at least one engineered recombination site comprising at leastone DNA sequence having at least 80-99% homology (or any range or valuetherein) to at least one of the above sequences, or any suitablerecombination site, or which hybridizes under stringent conditionsthereto, as known in the art.

[0248] Clearly, there are various types and permutations of suchwell-known in vitro and in vivo selection methods, each of which are notdescribed herein for the sake of brevity. However, such variations andpermutations are contemplated and considered to be the differentembodiments of the present invention.

[0249] It is important to note that as a result of the preferredembodiment being in vitro recombination reactions, non-biologicalmolecules such as PCR products can be manipulated via the presentrecombinational cloning method. In one example, it is possible to clonelinear molecules into circular vectors.

[0250] There are a number of applications for the present invention.These uses include, but are not limited to, changing vectors, apposingpromoters with genes, constructing genes for fusion proteins, changingcopy number, changing replicons, cloning into phages, and cloning, e.g.,PCR products, (with an attB site at one end and a loxP site at the otherend), genomic DNAs, and cDNAs.

[0251] Vector Donors

[0252] In accordance with the invention, any vector may be used toconstruct the Vector Donors of the invention. In particular, vectorsknown in the art and those commercially available (and variants orderivatives thereof) may in accordance with the invention be engineeredto include one or more recombination sites for use in the methods of theinvention. Such vectors may be obtained from, for example, VectorLaboratories Inc., InVitrogen, Promega, Novagen, NEB, Clontech,Boehringer Mannheim, Pharmacia, EpiCenter, OriGenes Technologies Inc.,Stratagene, Perkin Elmer, Pharmingen, Life Technologies, Inc., andResearch Genetics. Such vectors may then for example be used for cloningor subcloning nucleic acid molecules of interest. General classes ofvectors of particular interest include prokaryotic and/or eukaryoticcloning vectors, expression vectors, fusion vectors, two-hybrid orreverse two-hybrid vectors, shuttle vectors for use in different hosts,mutagenesis vectors, transcription vectors, vectors for receiving largeinserts and the like.

[0253] Other vectors of interest include viral origin vectors (M13vectors, bacterial phage λ vectors, adenovirus vectors, and retrovirusvectors), high, low and adjustable copy number vectors, vectors whichhave compatible replicons for use in combination in a single host(pACYC184 and pBR322) and eukaryotic episomal replication vectors(pCDM8).

[0254] Particular vectors of interest include prokaryotic expressionvectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B,and C, pRSET A, B, and C (Invitrogen, Inc.), pGEMEX-1, and pGEMEX-2(Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A, pKK223-3, thepGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc.), pKK233-2and pKK388-1 (Clontech, Inc.), and pProEx-HT (Life Technologies, Inc.)and variants and derivatives thereof. Vector donors can also be madefrom eukaryotic expression vectors such as pFastBac, pFastBac HT,pFastBac DUAL, pSFV, and pTet-Splice (Life Technologies, Inc.), pEUK-C1,pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo(Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.),p3′SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.),and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBsueBacIII, pCDM8,pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Inc.) andvariants or derivatives thereof

[0255] Other vectors of particular interest include pUC18, pUC19,pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificialchromosomes), BAC's (bacterial artificial chromosomes), P1 (E. coliphage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScript vectors,BlueScript vectors, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3(InVitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1(Life Technologies, Inc.) and variants or derivatives thereof

[0256] Additional vectors of interest include pTrxFus, pThioHis, pLEX,pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His,pcDNA3.1(−)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ,pPICZα, pGAPZ, pGAPZα, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5,pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1. pYES2, pZErO1.1, pZErO-2.1,pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1,pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe,SV2, pRc/CMV2, pRc/RSV, pREP4,pREP7, pREP8, pREP9, pREP10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni,and pCRBac from Invitrogen; λExCell, λ gt11, pTrc99A, pKK223-3,pGEX-1λT, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X,pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL,pMSG, pCH110, pKK232-8, pSL1 80, pNEO, and pUC4K from Pharmacia;pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg,pET-32 LIC, pET-30 LIC, pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC,pT7Blue-2, λSCREEN-1, λBlueSTAR, pET-3abcd, pET-7abc, pET9abcd,pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb,pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+),pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+),pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+),pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp, pBACgus-2cp,pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta—Hyg,and Selecta Vecta—Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1,pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1,pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6×His-GFP, pSEAP2-Basic,pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, pβgal-Basic,pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMVβ, pTet-Off, pTet-On,pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX,pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX 4T-1/2/3,pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTriplEx, λgt10,λgt11, pWE15, and λTriplEx from Clontech; Lambda ZAP II, pBK-CMV,pBK-RSV, pBluescript II KS±, pBluescript II SK±, pAD-GAL4, pBD-GAL4 Cam,pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4,SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS±, pBCKS±, pBC SK±, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc,pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS, pOPI3 CAT,pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, pOG45,pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414,pRS415, and pRS416 from Stratagene.

[0257] Two-hybrid and reverse two-hybrid vectors of particular interestinclude pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt,pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA,pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5,pNLexA, pYESTrp and variants or derivatives thereof.

[0258] Polymerases

[0259] Preferred polypeptides having reverse transcriptase activity(i.e., those polypeptides able to catalyze the synthesis of a DNAmolecule from an RNA template) include, but are not limited to MoloneyMurine Leukemia Virus (M-MLV) reverse transcriptase, Rous Sarcoma Virus(RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reversetranscriptase, Rous Associated Virus (RAV) reverse transcriptase,Myeloblastosis Associated Virus (MAV) reverse transcriptase, HumanImmunodeficiency Virus (HIV) reverse transcriptase, retroviral reversetranscriptase, retrotransposon reverse transcriptase, hepatitis Breverse transcriptase, cauliflower mosaic virus reverse transcriptaseand bacterial reverse transcriptase. Particularly preferred are thosepolypeptides having reverse transcriptase activity that are alsosubstantially reduced in RNAse H activity (i.e., “RNAse H-”polypeptides). By a polypeptide that is “substantially reduced in RNaseH activity” is meant that the polypeptide has less than about 20%, morepreferably less than about 15%, 10% or 5%, and most preferably less thanabout 2%, of the RNase H activity of a wildtype or RNase H⁺ enzyme suchas wildtype M-MLV reverse transcriptase. The RNase H activity may bedetermined by a variety of assays, such as those described, for example,in U.S. Pat. No. 5,244,797, in Kotewicz, M. L. et al., Nucl. Acids Res.16:265 (1988) and in Gerard, G. F., et al., FOCUS 14(5):91 (1992), thedisclosures of all of which are fully incorporated herein by reference.Suitable RNAse H⁻ polypeptides for use in the present invention include,but are not limited to, M-MLV H⁻ reverse transcriptase, RSV H⁻ reversetranscriptase, AMV H⁻ reverse transcriptase, RAV H⁻ reversetranscriptase, MAV H⁻ reverse transcriptase, HIV H⁻ reversetranscriptase, and SUPERSCRIPT™ I reverse transcriptase and SUPERSCRIPT™II reverse transcriptase which are available commercially, for examplefrom Life Technologies, Inc. (Rockville, Md.).

[0260] Other polypeptides having nucleic acid polymerase activitysuitable for use in the present methods include thermophilic DNApolymerases such as DNA polymerase I, DNA polymerase III, Klenowfragment, T7 polymerase, and T5 polymerase, and thermostable DNApolymerases including, but not limited to, Thermus thermophilus (Tth)DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoganeopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNApolymerase, Thermococcus litoralis (Tli or VENT®) DNA polymerase,Pyrococcus furiosus (Pfu or DEEPVENT®) DNA polymerase, Pyrococcus woosii(Pwo) DNA polymerase, Bacillus sterothermophilus (Bst) DNA polymerase,Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum(Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNA polymerase, Thermusruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME®) DNApolymerase, Methanobacterium thermoautotrophicum (Mth) DNA polymerase,and mutants, variants and derivatives thereof.

[0261] It will be understood by one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are readily apparent and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES

[0262] The present recombinational cloning method accomplishes theexchange of nucleic acid segments to render something useful to theuser, such as a change of cloning vectors. These segments must beflanked on both sides by recombination signals that are in the properorientation with respect to one another. In the examples below the twoparental nucleic acid molecules (e.g., plasmids) are called the InsertDonor and the Vector Donor. The Insert Donor contains a segment thatwill become joined to a new vector contributed by the Vector Donor. Therecombination intermediate(s) that contain(s) both starting molecules iscalled the Cointegrate(s). The second recombination event produces twodaughter molecules, called the Product (the desired new clone) and theByproduct.

[0263] Buffers

[0264] Various known buffers can be used in the reactions of the presentinvention. For restriction enzymes, it is advisable to use the buffersrecommended by the manufacturer. Alternative buffers can be readilyfound in the literature or can be devised by those of ordinary skill inthe art.

[0265] Examples 1-3. One exemplary buffer for lambda integrase iscomprised of 50 mM Tris-HCl, at pH 7.5-7.8, 70 mM KCl, 5 mM spermidine,0.5 mM EDTA, and 0.25 mg/ml bovine serum albumin, and optionally, 10%glycerol.

[0266] One preferred buffer for P1 Cre recombinase is comprised of 50 mMTris-HCl at pH 7.5, 33 mM NaCl, 5 mM spermidine, and 0.5 mg/ml bovineserum albumin.

[0267] The buffer for other site-specific recombinases which are similarto lambda Int and P1 Cre are either known in the art or can bedetermined empirically by the skilled artisans, particularly in light ofthe above-described buffers.

Example 1 Recombinational Cloning Using Cre and Cre & Int

[0268] Two pairs of plasmids were constructed to do the in vitrorecombinational cloning method in two different ways. One pair, pEZC705and pEZC726 (FIG. 2A), was constructed with loxP and att sites, to beused with Cre and λ integrase. The other pair, pEZC602 and pEZC629 (FIG.3A), contained the loxP (wild type) site for Cre, and a second mutantlox site, loxP 511, which differs from loxP in one base (out of 34total). The minimum requirement for recombinational cloning of thepresent invention is two recombination sites in each plasmid, in generalX and Y, and X′ and Y′. Recombinational cloning takes place if either orboth types of site can recombine to form a Cointegrate (e.g. X and X′),and if either or both can recombine to excise the Product and Byproductplasmids from the Cointegrate (e.g. Y and Y′). It is important that therecombination sites on the same plasmid do not recombine. It was foundthat the present recombinational cloning could be done with Cre alone.

[0269] Cre-Only

[0270] Two plasmids were constructed to demonstrate this conception (seeFIG. 3A). pEZC629 was the Vector Donor plasmid. It contained aconstitutive drug marker (chloramphenicol resistance), an origin ofreplication, loxP and loxP 511 sites, a conditional drug marker(kanamycin resistance whose expression is controlled by theoperator/promoter of the tetracycline resistance operon of transposonTn10), and a constitutively expressed gene for the tet repressorprotein, tetR. E. coli cells containing pEZC629 were resistant tochloramphenicol at 30 μg/ml, but sensitive to kanamycin at 100 μg/ml.pEZC602 was the Insert Donor plasmid, which contained a different drugmarker (ampicillin resistance), an origin, and loxP and loxP 511 sitesflanking a multiple cloning site.

[0271] This experiment was comprised of two parts as follows:

[0272] Part I: About 75 ng each of pEZC602 and pEZC629 were mixed in atotal volume of 30 μl of Cre buffer (50 mM Tris-HCl pH 7.5, 33 mM NaCl,5 mM spermidine-HCl, 500 μg/ml bovine serum albumin). Two 10 μl aliquotswere transferred to new tubes. One tube received 0.5 μl of Cre protein(approx. 4 units per μl; partially purified according to Abremski andHoess, J. Biol. Chem. 259:1509 (1984)). Both tubes were incubated at 37°C. for 30 minutes, then 70° C. for 10 minutes. Aliquots of each reactionwere diluted and transformed into DH5α. Following expression, aliquotswere plated on 30 μg/ml chloramphenicol; 100 μg/ml ampicillin plus 200μg/ml methicillin; or 100 μg/ml kanamycin. Results: See Table 1. Thereaction without Cre gave 1.11×10⁶ ampicillin resistant colonies (fromthe Insert Donor plasmid pEZC602); 7.8×10⁵ chloramphenicol resistantcolonies (from the Vector Donor plasmid pEZC629); and 140 kanamycinresistant colonies (background). The reaction with added Cre gave7.5×10⁵ ampicillin resistant colonies (from the Insert Donor plasmidpEZC602); 6.1×10⁵ chloramphenicol resistant colonies (from the VectorDonor plasmid pEZC629); and 760 kanamycin resistant colonies (mixture ofbackground colonies and colonies from the recombinational cloningProduct plasmid). Analysis: Because the number of colonies on thekanamycin plates was much higher in the presence of Cre, many or most ofthem were predicted to contain the desired Product plasmid. TABLE 1Enzyme Ampicillin Chloramphenicol Kanamycin Efficiency None 1.1 × 10⁶7.8 × 10⁵ 140 140/7.8 × 10⁵ = 0.02% Cre 7.5 × 10⁵ 6.1 × 10⁵ 760 760/6.1× 10⁵ = 0.12%

[0273] Part II: Twenty four colonies from the “+Cre” kanamycin plateswere picked and inoculated into medium containing 100 μg/ml kanamycin.Minipreps were done, and the miniprep DNAs, uncut or cut with SmaI orHindIII, were electrophoresed. Results: 19 of the 24 minipreps showedsupercoiled plasmid of the size predicted for the Product plasmid. All19 showed the predicted SmaI and HindIII restriction fragments.Analysis: The Cre only scheme was demonstrated. Specifically, it wasdetermined to have yielded about 70% (19 of 24) Product clones. Theefficiency was about 0.1% (760 kanamycin resistant clones resulted from6.1×10⁵ chloramphenicol resistant colonies).

[0274] Cre Plus Integrase

[0275] The plasmids used to demonstrate this method are exactlyanalogous to those used above, except that pEZC726, the Vector Donorplasmid, contained an attP site in place of loxP 511, and pEZC705, theInsert Donor plasmid, contained an attB site in place of loxP 511 (FIG.2A).

[0276] This experiment was comprised of three parts as follows:

[0277] Part I: About 500 ng of pEZC705 (the Insert Donor plasmid) wascut with ScaI, which linearized the plasmid within the ampicillinresistance gene. (This was done because the λ integrase reaction hasbeen historically done with the attB plasmid in a linear state (H. Nash,personal communication). However, it was found later that the integrasereaction proceeds well with both plasmids supercoiled.) Then, the linearplasmid was ethanol precipitated and dissolved in 20 μl of λ integrasebuffer (50 mM Tris-HCl, about pH 7.8, 70 mM KCl, 5 mM spermidine-HCl,0.5 mM EDTA, 250 μg/ml bovine serum albumin). Also, about 500 ng of theVector Donor plasmid pEZC726 was ethanol precipitated and dissolved in20 μl λ integrase buffer. Just before use, λ integrase (2 μl, 393 μg/ml)was thawed and diluted by adding 18 μl cold λ integrase buffer. One μlIHF (integration host factor, 2.4 mg/ml, an accessory protein) wasdiluted into 150 μl cold λ integrase buffer. Aliquots (2 μl) of each DNAwere mixed with λ integrase buffer, with or without 1 μl each λintegrase and IHF, in a total of 10 μl. The mixture was incubated at 25°C. for 45 minutes, then at 70° C. for 10 minutes. Half of each reactionwas applied to an agarose gel. Results: In the presence of integrase andIHF, about 5% of the total DNA was converted to a linear Cointegrateform. Analysis: Activity of integrase and IHF was confirmed.

[0278] Part II: Three microliters of each reaction (i.e., with orwithout integrase and IHF) were diluted into 27 μl of Cre buffer(above), then each reaction was split into two 10 μl aliquots (fouraltogether). To two of these reactions, 0.5 μl of Cre protein (above)were added, and all reactions were incubated at 37° C. for 30 minutes,then at 70° C. for 10 minutes. TE buffer (90 μl; TE: 10 mM Tris-HCl, pH7.5, 1 mM EDTA) was added to each reaction, and 1 μl each wastransformed into E. coli DH5α. The transformation mixtures were platedon 100 μg/ml ampicillin plus 200 μg/ml methicillin; 30 μg/mlchloramphenicol; or 100 μg/ml kanamycin. Results: See Table 2. TABLE 2Enzyme Ampicillin Chloramphenicol Kanamycin Efficiency None 990 20000 44/2 × 10⁴ = 0.02% Cre only 280 3640 0 0 Integrase* 1040 27000 9 9/2.7 ×10⁴ = only 0.03% Integrase* + 110 1110 76 76/ Cre 1.1 × 10³ = 6.9%

[0279] Analysis: The Cre protein impaired transformation. When adjustedfor this effect, the number of kanamycin resistant colonies, compared tothe control reactions, increased more than 100 fold when both Cre andIntegrase were used. This suggests a specificity of greater than 99%.

[0280] Part III: 38 colonies were picked from the Integrase plus Creplates, miniprep DNAs were made and cut with HindIII to give diagnosticmapping information. Result: All 38 had precisely the expected fragmentsizes. Analysis: The Cre plus λ integrase method was observed to havemuch higher specificity than Cre-alone. Conclusion: The Cre plus λintegrase method was demonstrated. Efficiency and specificity were muchhigher than for Cre only.

Example 2 Using in vitro Recombinational Cloning to Subclone theChloramphenicol Acetyl Transferase Gene into a Vector for Expression inEukaryotic Cells (FIG. 4A)

[0281] An Insert Donor plasmid, pEZC843, was constructed, comprising thechloramphenicol acetyl transferase gene of E. coli, cloned between loxPand attB sites such that the loxP site was positioned at the 5′-end ofthe gene (FIG. 4B). A Vector Donor plasmid, pEZC1003, was constructed,which contained the cytomegalovirus eukaryotic promoter apposed to aloxP site (FIG. 4C). One microliter aliquots of each supercoiled plasmid(about 50 ng crude miniprep DNA) were combined in a ten microliterreaction containing equal parts of lambda integrase buffer (50 mMTris-HCl, pH 7.8, 70 mM KCl, 5 mM spermidine, 0.5 mM EDTA, 0.25 mg/mlbovine serum albumin) and Cre recombinase buffer (50 mM Tris-HCl, pH7.5, 33 mM NaCl, 5 mM spermidine, 0.5 mg/ml bovine serum albumin), twounits of Cre recombinase, 16 ng integration host factor, and 32 nglambda integrase. After incubation at 30° C. for 30 minutes and 75° C.for 10 minutes, one microliter was transformed into competent E. colistrain DH5α (Life Technologies, Inc.). Aliquots of transformations werespread on agar plates containing 200 μg/ml kanamycin and incubated at37° C. overnight. An otherwise identical control reaction contained theVector Donor plasmid only. The plate receiving 10% of the controlreaction transformation gave one colony; the plate receiving 10% of therecombinational cloning reaction gave 144 colonies. These numberssuggested that greater than 99% of the recombinational cloning coloniescontained the desired product plasmid. Miniprep DNA made from sixrecombinational cloning colonies gave the predicted size plasmid (5026base pairs), CMVProd. Restriction digestion with NcoI gave the fragmentspredicted for the chloramphenicol acetyl transferase cloned downstreamof the CMV promoter for all six plasmids.

Example 3 Subcloned DNA Segments Flanked by attB Sites Without StopCodons

[0282] Part I: Background

[0283] The above examples are suitable for transcriptional fusions, inwhich transcription crosses recombination sites. However, both attR andloxP sites contain multiple stop codons on both strands, sotranslational fusions can be difficult, where the coding sequence mustcross the recombination sites, (only one reading frame is available oneach strand of loxP sites) or impossible (in attR or attL).

[0284] A principal reason for subcloning is to fuse protein domains. Forexample, fusion of the glutathione S-transferase (GST) domain to aprotein of interest allows the fusion protein to be purified by affinitychromatography on glutathione agarose (Pharmacia, Inc., 1995 catalog).If the protein of interest is fused to runs of consecutive histidines(for example His6), the fusion protein can be purified by affinitychromatography on chelating resins containing metal ions (Qiagen, Inc.).It is often desirable to compare amino terminal and carboxy terminalfusions for activity, solubility, stability, and the like.

[0285] The attB sites of the bacteriophage λ integration system wereexamined as an alternative to loxP sites, because they are small (25 bp)and have some sequence flexibility (Nash, H. A. et al., Proc. Natl.Acad. Sci. USA 84:4049-4053 (1987). It was not previously suggested thatmultiple mutations to remove all stop codes would result in usefulrecombination sites for recombinational subcloning.

[0286] Using standard nomenclature for site specific recombination inlambda bacteriophage (Weisber, in Lambda III, Hendrix, et al., eds.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)), thenucleotide regions that participate in the recombination reaction in anE. coli host cell are represented as follows:attP--P1--H1--P2--X--H2--C-O-C′--H′--P′1--P′2--P′3--                         + attB                 --B-O-B′--               Int, IHF ↓↑ Xis, Int, IHF attR--P1--H1--P2--X--H2--C-O-B′--                          +attL                 --B-O-C′--H′--P′1--P′2--P′3--,

[0287] where: O represents the 15 bp core DNA sequence found in both thephage and E. coli genomes; B and B′ represent approximately 5 basesadjacent to the core in the E. coli genome; and P1, H1, P2, X, H2, C,C′, H′, P′1, P′2, and P′3 represent known DNA sequences encoding proteinbinding domains in the bacteriophage λ genome.

[0288] The reaction is reversible in the presence of the protein Xis(excisionase); recombination between attL and attR precisely excise theλ genome from its integrated state, regenerating the circular λ genomecontaining attP and the linear E. coli genome containing attB.

[0289] Part II: Construction and Testing of Plasmids Containing Mutantatt Sites

[0290] Mutant attL and attR sites were constructed. Importantly, Landyet al. (Ann. Rev. Biochem. 58:913 (1989)) observed that deletion of theP1 and H1 domains of attP facilitated the excision reaction andeliminated the integration reaction, thereby making the excisionreaction irreversible. Therefore, as mutations were introduced in attR,the P1 and H1 domains were also deleted. attR′ sites in the presentexample lack the P1 and H1 regions and have the NdeI site removed (base27630 changed from C to G), and contain sequences corresponding tobacteriophage λ coordinates 27619-27738 (GenBank release 92.0, bg:LAMCG,“Complete Sequence of Bacteriophage Lambda”).

[0291] The sequence of attB produced by recombination of wild type attLand attR sites is: B O B′ attBwt: 5′ AGCCT GCTTTTTTATAC TAA CT TGA  3′(SEQ. ID NO: 60) 3′ TCGGA CGAAAA AAT AT GAT T GAACT 5′ (SEQ. ID NO: 44)

[0292] The stop codons are italicized and underlined. Note thatsequences of attL, attR, and attP can be derived from the attB sequenceand the boundaries of bacteriophage λ contained within attL and attR(coordinates 27619 to 27818).

[0293] When mutant attR1 (attR′) and attL1 sites were recombined thesequence attB1 was produced (mutations in bold, large font): B O B′attB1: 5′ AGCCT GCTTTTTT

TAC

AA CTTG

 3′ (SEQ. ID NO: 6) 3′ TCGGA CGAAAAAA

ATG

TT GAAC

 5′ (SEQ. ID NO: 45)

[0294] Note that the four stop codons are gone.

[0295] When an additional mutation was introduced in the attR1 (attR′)and attL1 sequences (bold), attR2 (attR′) and attL2 sites resulted.Recombination of attR2 and attL2 produced the attB2 site: B O B′ attB2:5′ AGCCT GCTTT

TTGTACAAA CTTGT 3′ (SEQ. ID NO: 7) 3′ TCGGA CGAAA

AACATGTTT GAACA 5′ (SEQ. ID NO: 46)

[0296] The recombination activities of the above attL and attR′ siteswere assayed as follows. The attB site of plasmid pEZC705 (FIG. 2B) wasreplaced with attLwt, attL1, or attL2. The attP site of plasmid pEZC726(FIG. 2C) was replaced with attRwt, attR1 (attR′, lacking regions P1 andH1) or attR2 (attR′, lacking regions P1 and H1). Thus, the resultingplasmids could recombine via their loxP sites, mediated by Cre, and viatheir attR′ and attL sites, mediated by Int, Xis, and IHF. Pairs ofplasmids were mixed and reacted with Cre, Int, Xis, and IHF, transformedinto E. coli competent cells, and plated on agar containing kanamycin.The results are presented in Table 3: TABLE 3 # of kanamycin Vectordonor att site Gene donor att site resistant colonies* attR'wt(pEZC1301) None  1 (background) ″ attLwt 147 ″ (pEZC1313)  47 ″ attL1(pEZC1317)  0 attL2 (pEZC1321) attR'1 (pEZC1305) None  1 (background) ″attLwt  4 ″ (pEZC1313) 128 ″ attL1 (pEZC1317)  0 attL2 (pEZC1321) attR'2(pEZC1309) None  0 (background) ″ attLwt  0 ″ (pEZC1313)  0 ″ attL1(pEZC1317) 209 attL2 (pEZC1321)

[0297] The above data show that whereas the wild type att and att1 sitesrecombine to a small extent, the att1 and att2 sites do not recombinedetectably with each other.

[0298] Part III. Recombination was demonstrated when the core region ofboth attB sites flanking the DNA segment of interest did not containstop codons. The physical state of the participating plasmids wasdiscovered to influence recombination efficiency.

[0299] The appropriate att sites were moved into pEZC705 and pEZC726 tomake the plasmids pEZC1405 (FIG. 5G) (attR′1 and attR′2) and pEZC1502(FIG. 5H) (attL1 and attL2). The desired DNA segment in this experimentwas a copy of the chloramphenicol resistance gene cloned between the twoattL sites of pEZC1502. Pairs of plasmids were recombined in vitro usingInt, Xis, and IHF (no Cre because no loxP sites were present). 100 ng ofeach plasmid were incubated in 10 μl reactions of 50 mM Tris HCl pHabout 7.8, 16.5 mM NaCl, 35 mM KCl, 5 mM spermidine, 0.25 mM EDTA, 0.375mg/ml BSA, 3% glycerol that contained 8.1 ng IHF, 43 ng Int, 4.3 ng Xis,and 2 units Cre. Reactions were incubated at 25° C. for 45 min., 65° C.for 10 min, and 1 μl aliquots were transformed into DH5α cells, andspread on kanamycin plates. The yield of desired kanamycin resistantcolonies was determined when both parental plasmids were circular, orwhen one plasmid was circular and the other linear as presented in Table4: TABLE 4 Vector donor¹ Insert donor¹ Kanamycin resistant colonies²Circular pEZC1405 None 30 Circular pEZC1405 Circular pEZC 1502 2680Linear pEZC 1405 None 90 Linear pEZC1405 Circular pEZC1502 172000Circular pEZC1405 Linear pEZC1502 73000

[0300] Analysis: Recombinational cloning using mutant attR and attLsites was confirmed. The desired DNA segment is subcloned between attBsites that do not contain any stop codons in either strand. The enhancedyield of Product DNA (when one parent was linear) was unexpected becauseof earlier observations that the excision reaction was more efficientwhen both participating molecules were supercoiled and proteins werelimiting (Nunes-Duby et al., Cell 50:779-788 (1987).

Example 4 Demonstration of Recombinational Cloning Without InvertedRepeats

[0301] Part I: Rationale

[0302] The above Example 3 showed that plasmids containing invertedrepeats of the appropriate recombination sites (for example, attL1 andattL2 in plasmid pEZC1502) (FIG. 5H) could recombine to give the desiredDNA segment flanked by attB sites without stop codons, also in invertedorientation. A concern was the in vivo and in vitro influence of theinverted repeats. For example, transcription of a desired DNA segmentflanked by attB sites in inverted orientation could yield a singlestranded RNA molecule that might form a hairpin structure, therebyinhibiting translation.

[0303] Inverted orientation of similar recombination sites can beavoided by placing the sites in direct repeat arrangement att sites. Ifparental plasmids each have a wild type attL and wild type attR site, indirect repeat the Int, Xis, and IHF proteins will simply remove the DNAsegment flanked by those sites in an intramolecular reaction. However,the mutant sites described in the above Example 3 suggested that itmight be possible to inhibit the intramolecular reaction while allowingthe intermolecular recombination to proceed as desired.

[0304] Part II: Structure of Plasmids Without Inverted Repeats forRecombinational Cloning

[0305] The attR2 sequence in plasmid pEZC1405 (FIG. 5G) was replacedwith attL2, in the opposite orientation, to make pEZC1603 (FIG. 6A). TheattL2 sequence of pEZC1502 (FIG. 5H) was replaced with attR2, in theopposite orientation, to make pEZC1706 (FIG. 6B). Each of these plasmidscontained mutations in the core region that make intramolecularreactions between att1 and att2 cores very inefficient (see Example 3,above).

[0306] Plasmids pEZC1405, pEZC1502, pEZC1603 and pEZC1706 were purifiedon Qiagen columns (Qiagen, Inc.). Aliquots of plasmids pEZC1405 andpEZC1603 were linearized with XbaI. Aliquots of plasmids pEZC1502 andpEZC1706 were linearized with AlwNI. One hundred ng of plasmids weremixed in buffer (50 mM Tris HCL pH about 7.8, 16.5 mM NaCl, 35 mM KCl, 5mM spermidine, 0.25 mM EDTA, 0.375 mg/ml BSA, 3% glycerol) containingInt (43.5 ng), Xis (4.3 ng) and IHF (8.1 ng) in a final volume of 10 μl.Reactions were incubated for 45 minutes at 25° C., 10 minutes at 65° C.,and 1 μl was transformed into E. coli DH5α. After expression, aliquotswere spread on agar plates containing 200 μg/ml kanamycin and incubatedat 37° C.

[0307] Results, expressed as the number of colonies per 1 μl ofrecombination reaction are presented in Table 5: TABLE 5 Vector DonorGene Donor Colonies Predicted % product Circular 1405 — 100 — Circular1405 Circular 1502 3740 3640/3740 = 97% Linear 1405 — 90 — Linear 1405Circular 1502 172,000 171,910/172,000 = 99.9% Circular 1405 Linear 150273,000 72,900/73,000 = 99.9% Circular 1603 — 80 — Circular 1603 Circular1706 410 330/410 = 80% Linear 1603 — 270 — Linear 1603 Circular 17067000 6730/7000 = 96% Circular 1603 Linear 1706 10,800 10,530/10,800 =97%

[0308] Analysis. In all configurations, i.e., circular or linear, thepEZC1405×pEZC1502 pair (with att sites in inverted repeat configuration)was more efficient than pEZC1603×pEZC1706 pair (with att sites mutatedto avoid hairpin formation). The pEZC1603×pEZC1706 pair gave higherbackgrounds and lower efficiencies than the pEZC1405×pEZC1502 pair.While less efficient, 80% or more of the colonies from thepEZC1603×pEZC1706 reactions were expected to contain the desired plasmidproduct. Making one partner linear stimulated the reactions in allcases.

[0309] Part III: Confirmation of Product Plasmids' Structure

[0310] Six colonies each from the linear pEZC1405 (FIG. 5G) x circularpEZC1502 (FIG. 5H), circular pEZC1405× linear pEZC1502, linear pEZC1603(FIG. 6A)×circular pEZC1706 (FIG. 6B), and circular pEZC1603× linearpEZC1706 reactions were picked into rich medium and miniprep DNAs wereprepared. Diagnostic cuts with Ssp I gave the predicted restrictionfragments for all 24 colonies.

[0311] Analysis. Recombination reactions between plasmids with mutantattL and attR sites on the same molecules gave the desired plasmidproducts with a high degree of specificity.

Example 5 Recombinational Cloning with a Toxic Gene

[0312] Part I: Background

[0313] Restriction enzyme DpnI recognizes the sequence GATC and cutsthat sequence only if the A is methylated by the dam methylase. Mostcommonly used E. coli strains are dam⁺. Expression of DpnI in dam⁺strains of E. coli is lethal because the chromosome of the cell ischopped into many pieces. However, in dam⁻ cells expression of DpnI isinnocuous because the chromosome is immune to DpnI cutting.

[0314] In the general recombinational cloning scheme, in which thevector donor contains two segments C and D separated by recombinationsites, selection for the desired product depends upon selection for thepresence of segment D, and the absence of segment C. In the originalExample segment D contained a drug resistance gene (Km) that wasnegatively controlled by a repressor gene found on segment C. When C waspresent, cells containing D were not resistant to kanamycin because theresistance gene was turned off.

[0315] The DpnI gene is an example of a toxic gene that can replace therepressor gene of the above embodiment. If segment C expresses the DpnIgene product, transforming plasmid CD into a dam⁺ host kills the cell.If segment D is transferred to a new plasmid, for example byrecombinational cloning, then selecting for the drug marker will besuccessful because the toxic gene is no longer present.

[0316] Part II: Construction of a Vector Donor Using DpnI as a ToxicGene

[0317] The gene encoding DpnI endonuclease was amplified by PCR usingprimers 5′CCA CCA CAA ACG CGT CCA TGG AAT TAC ACT TTA ATT TAG3′ (SEQ. IDNO: 17) and 5′CCA CCA CAA GTC GAC GCA TGC CGA CAG CCT TCC AAA TGT3′ (SEQID NO:18) and a plasmid containing the DpnI gene (derived from plasmidsobtained from Sanford A. Lacks, Brookhaven National Laboratory, Upton,N.Y.; also available from American Type Culture Collection as ATCC67494) as the template.

[0318] Additional mutations were introduced into the B and B′ regions ofattL and attR′, respectively, by amplifying existing attL and attR′domains with primers containing the desired base changes. Recombinationof the mutant attL3 (made with oligo Xis115) and attR′3 (attR′, madewith oligo Xis112) yielded attB3 with the following sequence(differences from attB1 in bold):   B           O         B′ ACCCAGCTTTCTTGTACAAA GTGGT (SEQ ID NO: 8) TGGGT CGAAAGAACATGTTT CACCA (SEQ IDNO: 47)

[0319] The attL3 sequence was cloned in place of attL2 of an existingGene Donor plasmid to give the plasmid pEZC2901 (FIG. 7A). The attR′3sequence was cloned in place of attR′2 in an existing Vector Donorplasmid to give plasmid pEZC2913 (FIG. 7B). The DpnI gene was clonedinto plasmid pEZC2913 to replace the tet repressor gene. The resultingVector Donor plasmid was named pEZC3101 (FIG. 7C). When pEZC3101 wastransformed into the dam⁻ strain SCS110 (Stratagene), hundreds ofcolonies resulted. When the same plasmid was transformed into the dam+strain DH5α, only one colony was produced, even though the DH5α cellswere about 20 fold more competent than the SCS110 cells. When a relatedplasmid that did not contain the DpnI gene was transformed into the sametwo cell lines, 28 colonies were produced from the SCS110 cells, while448 colonies resulted from the DH5α cells. This is evidence that the DpnI gene is being expressed on plasmid pEZC3101 (FIG. 7C), and that it iskilling the dam⁺ DH5α cells but not the dam⁻ SCS 110 cells.

[0320] Part III: Demonstration of Recombinational Cloning Using DpnISelection

[0321] A pair of plasmids was used to demonstrate recombinationalcloning with selection for Product dependent upon the toxic gene DpnI.Plasmid pEZC3101 (FIG. 7C) was linearized with MluI and reacted withcircular plasmid pEZC2901 (FIG. 7A). A second pair of plasmids usingselection based on control of drug resistance by a repressor gene wasused as a control: plasmid pEZC1802 (FIG. 7D) was linearized with XbaIand reacted with circular plasmid pEZC1502 (FIG. 5H). Eight microliterreactions containing buffer (50 mM Tris HCl pH about 7.8, 16.5 mM NaCl,35 mM KCl, 5 mM spermidine, 0.375 mg/ml BSA, 0.25 mM EDTA, 2.5%glycerol) and proteins Xis (2.9 ng), Int (29 ng), and IHF (5.4 ng) wereincubated for 45 minutes at 25° C., then 10 minutes at 75° C., and 1 μlaliquots were transformed into DH5α (i.e., dam+) competent cells, aspresented in Table 6. TABLE 6 Basis of Reaction # Vector donor selectionInsert donor Colonies 1 pEZC3101/Mlu Dpn I toxicity — 3 2 pEZC3101/MluDpn I toxicity Circular 4000 pEZC2901 3 pEZC1802/Xba Tet repressor — 0 4pEZC1802/Xba Tet repressor Circular 12100 pEZC1502

[0322] Miniprep DNAs were prepared from four colonies from reaction #2,and cut with restriction enzyme Ssp I. All gave the predicted fragments.

[0323] Analysis: Subcloning using selection with a toxic gene wasdemonstrated. Plasmids of the predicted structure were produced.

Example 6 Cloning of Genes with Uracil DNA Glycosylase and Subcloning ofthe Genes with Recombinational Cloning to Make Fusion Proteins

[0324] Part I: Converting an Existing Expression Vector to a VectorDonor for Recombinational Cloning

[0325] A cassette useful for converting existing vectors into functionalVector Donors was made as follows. Plasmid pEZC3101 (FIG. 7C) wasdigested with ApaI and KpnI, treated with T4 DNA polymerase and dNTPs torender the ends blunt, further digested with SmaI, HpaI, and AlwNI torender the undesirable DNA fragments small, and the 2.6 kb cassettecontaining the attR′1-Cm^(R)-Dpn I-attR′-3 domains was gel purified. Theconcentration of the purified cassette was estimated to be about 75 ngDNA/μl.

[0326] Plasmid pGEX-2TK (FIG. 8A) (Pharmacia) allows fusions between theprotein glutathione S transferase and any second coding sequence thatcan be inserted in frame in its multiple cloning site. pGEX-2TK DNA wasdigested with SmaI and treated with alkaline phosphatase. About 75 ng ofthe above purified DNA cassette was ligated with about 100 ng of thepGEX-2TK vector for 2.5 hours in a 5 μl ligation, then 1 μl wastransformed into competent E. coli BRL 3056 cells (a dam⁻ derivative ofDH10B; dam⁻ strains commercially available include DM1 from LifeTechnologies, Inc., and SCS 110 from Stratagene). Aliquots of thetransformation mixture were plated on LB agar containing 100 μg/mlampicillin (resistance gene present on pGEX-2TK) and 30 μg/mlchloramphenicol (resistance gene present on the DNA cassette). Colonieswere picked and miniprep DNAs were made. The orientation of the cassettein pGEX-2TK was determined by diagnostic cuts with EcoRI. A plasmid withthe desired orientation was named pEZC3501 (FIG. 8B).

[0327] Part II: Cloning Reporter Genes Into an Recombinational CloningGene Donor Plasmid in Three Reading Frames

[0328] Uracil DNA glycosylase (UDG) cloning is a method for cloning PCRamplification products into cloning vectors (U.S. Pat. No. 5,334,515,entirely incorporated herein by reference). Briefly, PCR amplificationof the desired DNA segment is performed with primers that contain uracilbases in place of thymidine bases in their 5′ ends. When such PCRproducts are incubated with the enzyme UDG, the uracil bases arespecifically removed. The loss of these bases weakens base pairing inthe ends of the PCR product DNA, and when incubated at a suitabletemperature (e.g., 37° C.), the ends of such products are largely singlestranded. If such incubations are done in the presence of linear cloningvectors containing protruding 3′ tails that are complementary to the 3′ends of the PCR products, base pairing efficiently anneals the PCRproducts to the cloning vector. When the annealed product is introducedinto E. coli cells by transformation, in vivo processes efficientlyconvert it into a recombinant plasmid.

[0329] UDG cloning vectors that enable cloning of any PCR product in allthree reading frames were prepared from pEZC3201 (FIG. 8K) as follows.Eight oligonucleotides were obtained from Life Technologies, Inc. (allwritten 5′→3′: rf1 top (GGCC GAT TAC GAT ATC CCA ACG ACC GAA AAC CTG TATTTT CAG GGT) (SEQ. ID NO:19), rf1 bottom (CAG GTT TTC GGT CGT TGG GATATC GTA ATC)(SEQ. ID NO:20), rf2 top (GGCCA GAT TAC GAT ATC CCA ACG ACCGAA AAC CTG TAT TTT CAG GGT)(SEQ. ID NO:21), rf2 bottom (CAG GTT TTC GGTCGT TGG GAT ATC GTA ATC T)(SEQ. ID NO:22), rf3 top (GGCCAA GAT TAC GATATC CCA ACG ACC GAA AAC CTG TAT TTT CAG GGT)(SEQ. ID NO:23), rf3 bottom(CAG GTT TTC GGT CGT TGG GAT ATC GTA ATC TT)(SEQ. ID NO:24), carboxy top(ACC GTT TAC GTG GAC)(SEQ. ID NO:25) and carboxy bottom (TCGA GTC CACGTA AAC GGT TCC CAC TTA TTA)(SEQ. ID NO:26). The rf1, 2, and 3 topstrands and the carboxy bottom strand were phosphorylated on their 5′ends with T4 polynucleotide kinase, and then the complementary strandsof each pair were hybridized. Plasmid pEZC3201 (FIG. 8K) was cut withNotI and SalI, and aliquots of cut plasmid were mixed with thecarboxy-oligo duplex (Sal I end) and either the rf1, rf2, or rf3duplexes (NotI ends) (10 μg cut plasmid (about 5 pmol) mixed with 250pmol carboxy oligo duplex, split into three 20 μl volumes, added 5 μl(250 pmol) of rf1, rf2, or rf3 duplex and 2 μl=2 units T4 DNA ligase toeach reaction). After 90 minutes of ligation at room temperature, eachreaction was applied to a preparative agarose gel and the 2.1 kb vectorbands were eluted and dissolved in 50 μl of TE.

[0330] Part III: PCR of CAT and phoA Genes

[0331] Primers were obtained from Life Technologies, Inc., to amplifythe chloramphenicol acetyl transferase (CAT) gene from plasmid pACYC184,and phoA, the alkaline phosphatase gene from E. coli. The primers had12-base 5′ extensions containing uracil bases, so that treatment of PCRproducts with uracil DNA glycosylase (UDG) would weaken base pairing ateach end of the DNAs and allow the 3′ strands to anneal with theprotruding 3′ ends of the rf1, 2, and 3 vectors described above. Thesequences of the primers (all written 5′→3′) were: CAT left, UAU UUU CAGGGU ATG GAG AAA AAA ATC ACT GGA TAT ACC (SEQ. ID NO:27); CAT right, UCCCAC UUA UUA CGC CCC GCC CTG CCA CTC ATC (SEQ. ID NO:28); phoA left, UAUUUU CAG GGU ATG CCT GTT CTG GAA AAC CGG (SEQ. ID NO:29); and phoA right,UCC CAC UUA UUA TTT CAG CCC CAG GGC GGC TTT C (SEQ. ID NO:30). Theprimers were then used for PCR reactions using known method steps (see,e.g., U.S. Pat. No. 5,334,515, entirely incorporated herein byreference), and the polymerase chain reaction amplification productsobtained with these primers comprised the CAT or phoA genes with theinitiating ATGs but without any transcriptional signals. In addition,the uracil-containing sequences on the amino termini encoded thecleavage site for TEV protease (Life Technologies, Inc.), and those onthe carboxy terminal encoded consecutive TAA nonsense codons.

[0332] Unpurified PCR products (about 30 ng) were mixed with the gelpurified, linear rf1, rf2, or rf3 cloning vectors (about 50 ng) in a 10μl reaction containing 1× REact 4 buffer (LTI) and 1 unit UDG (LTI).After 30 minutes at 37° C., 1 μl aliquots of each reaction weretransformed into competent E. coli DH5α cells (LTI) and plated on agarcontaining 50 μg/ml kanamycin. Colonies were picked and analysis ofminiprep DNA showed that the CAT gene had been cloned in reading frame 1(pEZC3601) (FIG. 8C), reading frame 2 (pEZC3609) (FIG. 8D) and readingframe 3 (pEZC3617) (FIG. 8E), and that the phoA gene had been cloned inreading frame 1 (pEZC3606) (FIG. 8F), reading frame 2 (pEZC3613) (FIG.8G) and reading frame 3 (pEZC3621) (FIG. 8H).

[0333] Part IV: Subcloning of CAT or phoA from UDG Cloning Vectors intoa GST Fusion Vector

[0334] Plasmids encoding fusions between GST and either CAT or phoA inall three reading frames were constructed by recombinational cloning asfollows. Miniprep DNA of GST vector donor pEZC3501(FIG. 8B) (derivedfrom Pharmacia plasmid pGEX-2TK as described above) was linearized withClaI. About 5 ng of vector donor were mixed with about 10 ng each of theappropriate circular gene donor vectors containing CAT or phoA in 8 μlreactions containing buffer and recombination proteins Int, Xis, and IHF(Example 5). After incubation, 1 μl of each reaction was transformedinto E. coli strain DH5α and plated on ampicillin, as presented in Table7. TABLE 7 Colonies (10% of DNA each transformation) Linear vector donor(pEZC3501/Cla) 0 Vector donor + CAT rf1 110 Vector donor + CAT rf2 71Vector donor + CAT rf3 148 Vector donor + phoA rf1 121 Vector donor +phoA rf2 128 Vector donor + phoA rf3 31

[0335] Part V: Expression of Fusion Proteins

[0336] Two colonies from each transformation were picked into 2 ml ofrich medium (CircleGrow, Bio101 Inc.) in 17×100 mm plastic tubes (Falcon2059, Becton Dickinson) containing 100 μg/ml ampicillin and shakenvigorously for about 4 hours at 37° C., at which time the cultures werevisibly turbid. One ml of each culture was transferred to a new tubecontaining 10 μl of 10% (w/v) IPTG to induce expression of GST. After 2hours additional incubation, all cultures had about the same turbidity;the A600 of one culture was 1.5. Cells from 0.35 ml each culture wereharvested and treated with sample buffer (containing SDS andβ-mercaptoethanol) and aliquots equivalent to about 0.15 A600 units ofcells were applied to a Novex 4-20% gradient polyacrylamide gel.Following electrophoresis the gel was stained with Coomassie blue.

[0337] Results: Enhanced expression of single protein bands was seen forall 12 cultures. The observed sizes of these proteins correlated wellwith the sizes predicted for GST being fused (through attB recombinationsites without stop codons) to CAT (FIG. 8I) or phoA (FIG. 8J) in threereading frames: CAT rf1=269 amino acids; CAT rf2=303 amino acids; CATrf3=478 amino acids; phoA rf1=282 amino acids; phoA rf2=280 amino acids;and phoA rf3=705 amino acids.

[0338] Analysis: Both CAT and phoA genes were subcloned into a GSTfusion vector in all three reading frames, and expression of the sixfusion proteins was demonstrated.

Example 7 Reverse Recombination and Subcloning by Recombination

[0339] Two plasmids were constructed to demonstrate reverserecombination according to the present invention. The vector pEZC5601(FIG. 10A), containing attB recombination sites and termed the attBparent plasmid (this vector may correspond to the Product DNA), furthercontained an ampicillin resistance gene, an origin of replication, anattB2 site, a tetracycline resistance gene, and an attB0 site, asdescribed above. Plasmid pEZC6701 (FIG. 10B), containing attPrecombination sites and termed the attP parent plasmid (this vector maycorrespond to the Byproduct DNA or may correspond to a different VectorDonor DNA), also contained a kanamycin resistance gene, an origin ofreplication, an attP2 site, a gene encoding the toxic protein ccdB, andan attP0 site. Integrase buffer at 10× concentration comprised 0.25 MTris HCl pH 7.5, 0.25 M Tris HCl pH 8.0, 0.7 M potassium chloride, 50 mMspermidine HCl, 5mM EDTA, and 2.5 mg/ml BSA. Note that attP0 and attP2contained the P1 and H1 domains. Integrase (1.5 μl of 435 ng/μl) and IHF(1.5 μl of 16 ng/μl in 1× Integrase buffer) were mixed with 13.5 μl of1× Int buffer to make the recombinase mixture.

[0340] Two 8 μl reactions were assembled. Reaction A contained 300 ngpEZC6701 plasmid and 2 μl of recombinase mixture in 1× Integrase buffer.Reaction B contained 300 ng pEZC5601, 300 ng pEZC6701, and 2 μl ofrecombinase mixture in 1× Integrase buffer. Both reactions wereincubated at 25° C. for 45 minutes, then at 70° C. for 5 minutes, andthen cooled. TE buffer (792 μl of 10 mM Tris HCl pH 7.5, 1 mM EDTA) wasadded to each reaction, and 1 μl of this diluted reaction wastransformed into DH5α UltraMax competent E. coli cells (LifeTechnologies, Inc., Rockville, Md.). After 1 hour of expression innon-selective medium, one tenth (100 μl) of each transformation wasspread onto agar plates containing 100 μg/ml kanamycin.

[0341] After overnight incubation at 37° C., the plate from reaction Acontained 1 colony, while the plate from reaction B contained 392colonies. Twelve colonies were picked from the reaction B plate intorich liquid medium and grown overnight. Miniprep DNAs prepared fromthese cultures were run uncut on an agarose gel and all 12 contained aplasmid of about 3.8 kb. Six of the miniprep DNAs were cut withrestriction enzyme ClaI and run along with pEZC6701 (the kanamycinresistant parental plasmid) also cut with ClaI. Plasmid pEZC6701 was cutonce with ClaI to give a fragment of about 3.8 kb. The six miniprep DNAscut twice with ClaI to give fragments of about 900 base pairs and about2900 base pairs.

[0342] Analysis: Recombination between the attP sites on pEZC6701 andthe attB sites on pEZC5601 resulted in the production of two daughterplasmids, the attL product plasmid (FIG. 10C) (which may correspond tothe Vector Donor DNA or a new Byproduct DNA) that contained theampicillin resistance and ccdB genes, and the attR product plasmid (FIG.10D) (which may also correspond to the Insert Donor DNA or a new ProductDNA) that contained the kanamycin and tetracycline resistance genes.Competent E. coli cells that received the attL product plasmid, the attPparent plasmid pEZC6701, or recombination intermediates, were killed bythe toxic ccdB gene product. Competent E. coli cells that received theattB parent plasmid pEZC5601 were killed by the kanamycin selection.Only competent E. coli cells that received the desired attR productplasmid, comprising the kanamycin and tetracycline resistance genes,survived to form colonies. The success of the selection strategy wasindicated by the large number of colonies from the reaction thatcontained both parental plasmids, compared to the reaction thatcontained only one parental plasmid. The reaction mechanism predictedthat the desired product plasmid would contain two ClaI restrictionsites, one in the kanamycin resistance gene from the pEZC6701 attPparent plasmid and one in the tetracycline resistance gene from thepEZC5601 attB parent plasmid. The presence of the two sites and thesizes of the fragments resulting from the ClaI digestion confirmed thereaction mechanism.

[0343] Thus, the present invention relates to reversal of therecombination reaction shown in FIG. 1, in which the Product DNA andByproduct DNA may be combined to produce the Insert Donor DNA and theVector Donor DNA. Additionally, the invention provides for subcloningrecombinations, in which a Product DNA (produced according to FIG. 1)may be combined with a new Vector Donor DNA to produce a new Product DNA(in a different Vector background) and a new Byproduct.

Example 8 Subcloning of Linearized Fragments

[0344] Plasmid pEZC7102 (FIG. 11A), the attP parent plasmid (which maycorrespond to the Vector Donor DNA), contained segments attP1, origin ofreplication, kanamycin resistance, attP3, chloramphenicol resistance,and the toxic gene ccdB, and in the experiment described here wassupercoiled. Plasmid pEZC7501 (FIG. 11B), the attB parent plasmid (whichmay correspond to the Insert Donor DNA or the Product DNA), containedthe GFP gene cloned between attB 1 and attB3 sites in a vector thatcomprised the functional domains of pCMVSPORT2.0 (Life Technologies,Inc.). The attP sites contained the P1 and H1 domains. Plasmid pEZC7501was used uncut, or was linearized within the ampicillin resistance genewith ScaI, or was cut with XbaI and SalI, to yield a fragment comprisingthe SalI end, 22 bp, the attB1 site, the GFP gene, the attB3 site, and14 bp to the XbaI end:

[0345] SalI end—22 bp—attB1—GFP—attB3—14 bp—XbaI end

[0346] Reactions (8 μl final volume) contained about 40 ng of each DNA,1× Int buffer (25 mM Tris HCl pH 7.5, 25 mM Tris HCl pH 8.0, 70 mM KCl,5 mM spermidine HCl, 0.5 mM EDTA, and 0.25 mg/ml BSA), 12.5% glycerol, 8ng IHF, and 43 ng lambda integrase. Reactions were incubated at 25° C.for 45 minutes, then at 70° C. for 5 minutes, and then cooled. Duplicate1 μl aliquots of each reaction were transformed into DH5α UltraMax cellsand plated in duplicate on kanamycin agar plates.

[0347] The reaction that contained only (supercoiled) pEZC7102 gave anaverage of 2 colonies (range 1 to 4). The reaction that contained bothpEZC7102 and supercoiled pEZC7501 gave an average of 612 colonies (range482-762). The reaction that contained pEZC7102 and linear (ScaI-cut)pEZC7501 gave an average of 360 colonies (range 127-605). The reactionthat contained pEZC7102 and the GFP gene on a fragment with attB sitesand 22 bp and 14 bp beyond the attB sites (pEZC7501 cut with SalI andXbaI) gave an average of 274 colonies (range 243-308).

[0348] Miniprep DNAs were prepared from 4 colonies from the pEZC7102×supercoiled pEZC7510 reaction, and from 10 colonies from the pEZC7102×pEZC7501/SalI+XbaI reaction. All 14 DNAs were run uncut on an agarosegel, and the 10 DNAs from the pEZC7102×pEZC7501/SalI+XbaI reaction werecut with a mixture of NcoI and PstI and run on an agarose gel. All theuncut plasmids were about 2.8 kb in size. All ten plasmids cut with themixture of NcoI and PstI gave fragments of about 700 and 2100 bp.

[0349] The results are presented in Table 8: TABLE 8 Colonies Uncut(average product attP attB of 4 Minipreps plasmid Fragment sizes, ParentParent plates) done size Nco + Pst digest sc 7102 — 2 — — — sc 7102 sc7501 612  4 2.8 kb — sc 7102 7501/ScaI 360 — — — sc 7102 7501/SalI +XbaI 274 10 2.8 kb ca. 2100 bp, 700 bp

[0350] Analysis: It was expected that the integrative reaction betweenthe attB sites on plasmid pEZC7501 and the attP sites on plasmidpEZC7102 would produce the attL product plasmid (FIG. 11C)(corresponding to the Insert Donor DNA) containing the GFP segment frompEZC7501, and the kanamycin—origin segment from pEZC7102. The presenceof the toxic gene ccdB on the attP parent plasmid pEZC7102(corresponding to the Byproduct DNA) was predicted to kill all the cellsthat received this plasmid. The large increase in the number of colonieswhen pEZC7501 was present indicated that the desired reaction wasoccurring, and that the efficiency of the reaction was adequate even ifthe attB parent plasmid (corresponding to the Product DNA) was linear(ScaI cut), or if the attB sites and the GFP gene were present an afragment that contained little additional sequence beyond the attBsites.

[0351] These results show that linear fragments can be suitablysubcloned into a different vector by the method of the invention.

Example 9 Cloning Long PCR Fragments

[0352] A PCR product was designed to have an attB0 (wild type) site atone end and a loxP site at the other end. The rationale was that theattP0×attB0 reaction would go well with the attB0 molecule (the PCRproduct) linear, (since it involves a normal lambda integrationreaction), and that the loxP×loxP excision from the cointegrate wouldalso be efficient (the unimolecular excision reaction is efficient, thebimolecular integration reaction is inefficient with Cre).

[0353] The sequence of the attB-containing PCR primer was 5_-TCC GTT GAAGCC TGC TTT TTT ATA CTA ACT TGA GCG AAG CCT CGG GGT CAG CAT AAG G-3′(SEQ ID NO:31). The sequence of the loxP primer was 5′-CCA ATA ACT TCGTAT AGC ATA CAT TAT ACG AAG TTA TTG CCC CTT GGT GAC ATA CTC G-3′ (SEQ IDNO:32). These primers amplify a part of the human myosin heavy chain.Polymerase chain reactions were performed using ELONGASE™ and K562 humanDNA as template. Polymerase chain reactions were performed as follows.Reactions (50 microliters) contained 100 ng K562 human DNA (LifeTechnologies, Inc.), 0.2 μM of each primer, and 0.2 mM of each dNTP, inELONGASE™ SuperMix (Life Technologies, Inc.). Reactions in thin walltubes under mineral oil were denatured at 94° C. for 1 minute, thencycled 35 times at 94_C. for 30 seconds, 65° C. for 30 seconds, and 68°C. for 8 minutes 30 seconds. Following thermal cycling, reactions weremaintained at 4° C. The 5.2 kb PCR product (FIG. 9A) was gel purified.

[0354] Plasmid pEZC1202 (FIG. 9B) contained a wild-type attP site, achloramphenicol resistance gene, a gene encoding the tet repressor, awild-type loxP site, an origin of replication, and a tetoperator/promoter transcribing and controlling the transcription of akanamycin resistance gene. This plasmid conferred chloramphenicolresistance but not kanamycin resistance, because the tet repressor madeby one element of the plasmid kept the kanamycin resistance gene turnedoff. The pEZC1202 DNA used in this experiment was a miniprep whoseconcentration was estimated to be about 50 ng per microliter.

[0355] About 40 ng of the gel purified 5.2 kb PCR product were includedin a 10 μl reaction that contained about 50 ng of supercoiled pEZC1202,0.2 units of Cre recombinase, 3.6 ng IHF, and 11 ng of Int in 50 mM TrisHCl pH about 7.8, 16 mM NaCl, 35 mM KCl, 0.25 mM EDTA, 0.375 mg/mlbovine serum albumin. A second reaction that did not contain the PCRproduct was included as a control. After incubating at 27° for 45 minand then 70° for 5 minutes, 1 μl aliquots were transformed into DH5αUltraMax competent E. coli cells (Life Technologies, Inc.). One fifth ofeach expression mix was plated on agar that contained 100 μg/mlkanamycin and the plates were incubated overnight at 37° C. The reactionthat contained the PCR product gave 34 colonies, while the reaction thatlacked the PCR product gave 31 colonies. After the plates sat at roomtemperature for four days, 26 additional small colonies were seen on theplate from the positive (+PCR product) reaction, while only oneadditional small colony was seen on the plate from the negative (no PCRproduct) reaction.

[0356] Twelve of the 26 small colonies were grown overnight in richbroth (CircleGrow) that contained 25 μg/ml kanamycin, and miniprep DNAswere prepared from these cultures. All twelve miniprep plasmids wereabout 8 kb in size, which corresponded to the size expected forreplacement of the choramphenicol resistance and tet repressor genes inpEZC1202 with the 5.2 kb PCR product. The predicted recombinant productis shown in FIG. 9C. Two of these plasmids were cut with AvaI (8 sitespredicted) and BamHI (4 sites predicted). All the predicted AvaIfragments appeared to be present. One of the BamH I sites predicted inthe PCR product (the one closest to the attB end) was absent from bothminipreps, but the other BamHI fragments were consistent with theexpected structure of the cloned 5.2 kb PCR product.

[0357] Analysis: The replacement of the choramphenicol resistance andtet repressor genes in pEZC1202 with the 5.2 kb PCR product (part of thehuman myosin heavy chain) conferred a moderate resistance of the host E.coli cells to kanamycin, but this resistance was not sufficient to allowcolonies to appear after overnight incubation. Thus, colonies containingthe desired recombination product grew on kanamycin plates, but were notseen after overnight incubation, but only after an additional roomtemperature incubation. Of the 12 AvaI and BamHI restriction sitespredicted from the nucleotide sequence, 11 were confirmedexperimentally. Thus the following three observations support theconclusion that the 5.2 kb PCR product was cloned by recombination: (a)small, slow growing colonies appeared only on the plate from thereaction that contained the PCR product; (b) the miniprep plasmids fromthese colonies were the expected size; and (c) diagnostic restrictioncuts gave the expected fragments (with the one above noted exception).

Example 10 Cloning of PCR Fragments

[0358] Three sets of pairs of PCR primers (Table 9) were designed toamplify an 830 bp sequence within plasmid pEZC7501 (FIG. 11B)comprising: attB 1—GFP—attB3, with or without additional nucleotides atthe outer ends of the 25 bp attB1 and attB3 recombination sites. (Here“outer” refers to the end of the attB sequence that is not adjacent tothe GFP gene sequence.) Primer set A added 17 nucleotides upstream ofattB1 and 15 nucleotides downstream of attB3; primer set B added 5 and 8nucleotides to attB1 and attB3, respectively; and primer set C added noadditional nucleotides to either attB recombination sequence.

[0359] The primer sequences are provided in Table 9: TABLE 9 upper GFP A5′-TCA CTA GTC GGC GGC CCA CA (SEQ ID NO: 33) lower GFP A 5′-GAG CGG CCCCCG CGG ACC AC (SEQ ID NO: 34) upper GFP B 5′-GGC CCA CAA GTT TGT ACAAAA (SEQ ID NO: 35) lower GFP B 5′-CCC CGC GGA CCA CTT TGT AC (SEQ IDNO: 36) upper GFP C 5′-ACA AGT TTG TAC AAA AAA GCA (SEQ ID NO: 37) lowerGFP C 5′-ACC ACT TTG TAC AAG AAA GCT (SEQ ID NO: 38)

[0360] PCR Reactions

[0361] Primer sets A and C were used first with the following PCRreactions, in 50 μl, in duplicate. Final concentrations were:

[0362] 20 mM TrisHCl, pH 8.4

[0363]50 mM KC1

[0364] 0.2 mM of all four deoxynucleotide triphosphates (dNTPs)

[0365] 400 ng/ml pEZC7501 supercoiled DNA template

[0366] 0.5 μM of each primer

[0367] Recombinant Taq DNA polymerase (BRL-GIBCO) 100 U/ml

[0368] A duplicate set of the above reactions contained 1 M betaine.

[0369] The reactions were first heated for to 94° C. for 1′, then cycled25 times at 94° C. for 45″, 55° C. for 30″, and 72° C. for 1′.

[0370] The size of the PCR reaction products was analyzed on a 1%agarose gel in TAE buffer containing 0.5 μg/ml ethidium bromide. Allreactions yielded products of the expected size, thus duplicatereactions were pooled. As the corresponding reactions with and withoutbetaine were not significantly different, these also were pooled, givinga final pooled volume for reactions with primer sets A and C of 200 μleach.

[0371] Primer set B was then used with identical reactions to thoseabove performed, except that the reaction volumes were increased to 100μl. After duplicate reactions and reactions plus and minus betaine werepooled, the final volume of the reactions with primer set B was 400 μl.

[0372] The three pooled primer reaction products were stored at −20° C.for 4 weeks.

[0373] PCR Product Purification

[0374] Each of the three pooled PCR products was extracted once with anequal volume of a mixture of Tris-buffered phenol, isoamyl alcohol andchloroform. The aqueous supernatant then was extracted twice with anequal volume of isobutanol, and the aqueous layer ethanol precipitatedwith two volumes of ethanol, 0.1 M sodium acetate, pH 6.2. The ethanolprecipitates were recovered by centrifugation at 13,000 rpm for 10′ atroom temperature, and the supernatant discarded. The dried pellets weredissolved in TE: 100 μl for reactions prepared with primer sets A and C;200 μl for the reactions with primer set B.

[0375] To remove PCR primers and extraneous small PCR products, the PCRproducts were precipitated with polyethylene glycol (PEG) by adding ½volume of a solution of 30% PEG 8000 (Sigma), 30 mM MgCl₂, mixing well,and centrifuging at 13,000 rpm for 10′, all at room temperature. Thesupernatant was discarded, and the pellets were dissolved in theirprevious volume of TE buffer. 1 μl aliquots of each of the three PCRproducts were checked on a 1% agarose gel to quantitate the recovery,which was estimated to be over 90%. The concentration of each PCRproduct was adjusted using TE to 40 ng/μl.

[0376] Recombination Reaction with the PCR Products of Primer sets A, B,and C

[0377] Five 8 μl reactions were assembled in 1× Integrase buffer (25 mMTris HCl pH 7.5, 25 mM Tris HCl pH 8.0, 80 mM KCl, 5 mM spermidine, 0.5mM EDTA, 0.25 mg/ml BSA) containing: 40 ng of pEZC7102 DNA, 2 □l ofrecombinase mixture (8 ng/μl IHF, 22 ng/μl Int in 1× Int Buffer, 50%glycerol) the reactions differed by the addition of either the PCRproduct of primer set A (reaction A), primer set B (reaction B), orprimer set C (reaction C); the addition of no PCR product (reaction D),or the addition of 40 ng of pEZC7501 SC (supercoiled) DNA(reaction E) asa positive control. All reactions were performed in duplicate.

[0378] The reactions were incubated for 45′ at 25° C., for 10′ at 70°C., then held at 0-5° C. 2 μl aliquots of each reaction were transformedinto Max Efficiency DH5α, in a 50 μl transformation reaction, andfollowing expression in 50C medium, ⅕ (100 μl) and ⅘ (400 μl) of thereactions were plated on kanamycin-containing (50 μg/ml) LB cultureplates. The results of the duplicate reactions are shown in Table 10.TABLE 10 Transfection No. Colonies A 100 μl 464, 668 A 400μl >1000, >1300 B 100 μl 980, 1292 B 400 μl >3000, >3000 C 100 μl 2, 8 C400 μl 13, 20 D 100 μl 0, 0 D 400 μl 0, 0 E 100 μl 56, 70

[0379] Analysis of the Colonies Obtained

[0380] Miniprep DNA was prepared from 8 colonies of each of theRecombination reactions with primer sets A, B. or C. The supercoiled DNAobtained was analyzed on a 1% agarose gel: all eight of colonies fromthe recombination products of primer sets A and B were of the predictedsize (2791 bp) for correct recombination between the PCR products (about824 bp) and the attB1—ori—kan^(r)—attB3 sequence donated by pEZC 7102(1967 bp). Three of the eight reaction products of primer set C were ofthe predicted size; the other five all were slightly larger than 4 kb.

[0381] Further analysis of the reaction products was performed using twodifferent restriction enzymes, AvaI and PvuII, each of which cleavestwice (but at different locations) within the predicted recombinantproduct, once within the PCR product sequence and once within thesequence contributed by pEZC7102. Both of these enzymes should cleavethe intact pEZC7102 recombination partner plasmid at two sites, to givefragments easily distinguished from those of the expected recombinationproducts.

[0382] The two restriction enzyme digests yielded the expected sizes offragments (2373 and 430 bp for AvaI; 2473 and 330 bp for PvuII) from thecolonies generated from the recombination reactions with primer sets Aand B, as well as for the three colonies from primer set C thatdisplayed the expected size of supercoiled DNA. For the other fivecolonies from primer set C that yielded larger SC DNA, however, thePvuII digest revealed fragments of approximate size to those predictedfrom a digestion of pEZC7102, whereas the AvaI digest revealed only asingle fragment, approximately the size of linearized pEZC7102 (4161bp).

[0383] Analysis

[0384] These results indicate that PCR products generated from templatescontaining a gene flanked by attB sites can serve as efficientsubstrates for the reverse recombination reaction. The addition of evenshort DNA sequences to the ends of the attB1 and attB3 sites or coreregions (e.g., 5 bp and 8 bp, respectively, in primer set B) stimulatedthis reaction by 100 fold or more. Surprisingly, reverse recombinationreactions with PCR products containing additional sequence beyond theattB sites appeared in these reactions to be more efficientrecombination partners than the supercoiled positive control plasmid,pEZC7501.

[0385] All the recombination products were generated faithfully. A lowlevel of background colonies emerged from the relatively inefficientrecombination reactions with primer set C, which lacked additionalsequence beyond the 25 bp attB sites. This background appeared to be dueto a largely intact pEZC7102 (which encodes kanamycin resistance)lacking an active ccdB death gene, allowing it to survive. Consistentwith this interpretation is that one of the two restriction sites forAvaI in this plasmid was also altered. One of the AvaI sites is presentwithin the ccdB region of pEZC7102. It is likely therefore that thealteration of this site was secondary to mutational inactivation of theccdB gene.

Example 11 Further Cloning of PCR Fragments

[0386] Two sets of 6 primers for preparing PCR products from the plasmidpBR322 as template were used. One set (Table 11) anneals to sequencesflanking the TetR gene, including the TetR promoter. The other set(Table 12) anneals to sequences flanking the AmpR gene, including itspromoter. The “tet” and “amp” primers used contain no attB sequences,only sequences inherent to the pBR322 plasmid; the “attB” primerscontain, in addition to the pBR322 sequences, the 25 bp of attB 1 orattB3 sequences; the “attB+4” primers contain the pBR322-specificsequences, plus the 25 bp attB 1 or attB3 sequences, each with four Gsat the outer end. (Here “outer” refers to the end of the attB sequencenot adjacent to the template-specific primer sequence.)

[0387] Preparation of pBR322 Template

[0388] To improve the efficiency of the PCR reaction, the supercoiledpBR322 DNA was linearized by incubating 3.5 μg of Suerpcoiled (SC)pBR322 DNA in a 200 μl reaction with 15 units of the restriction enzymeNdeI and final concentration of 50 mM Tris-HCl, pH8.0, 10 mM MgCl₂, and50 mM NaCl, for one hour at 37° C.

[0389] The digested pBR322 DNA was extracted once with phenol, isoamylalcohol, and chloroform, extracted twice with isobutanol, andprecipitated by adding two volumes of ethanol plus 0.15M sodium acetate.The precipitate was washed once with 100% ethanol, dried, then dissolvedin TE buffer. Recovery of DNA, quantitated on a 1% agarose gel in TAEbuffer, 0.5 □g/ml ethdium bromide, was estimated as greater than 80%.TABLE 11 tet SEQ Primer Primer Sequence ID NO: tet-L AAT TCT CAT GTT TGACAG CTT 48 ATC tet-R CGA TGG ATA TGT TCT GCC AAG 49 attB1- ACAAG TTTGTACAAAAA AGCA GGCTAAT 50 tetL TCT CAT GTT TGA CAG CTT ATC attB3- ACCACTTTGTA CAAGAA AGCT GGGTCGA 51 tetR TGG ATA TGT TCT GCC AAG attB1 + GGGGACAAG TTTGTA CAAAAA AGCAGGCT 52 4-tetL AAT TCT CAT GTT TGA GAG CTTATCattB3 + GGGG ACCAC TTTGTA CAAGAA AGCTGGGT 53 4-tetR CGA TGG ATA TGT TCTGCC AAG

[0390] TABLE 12 tet SEQ Primer Primer Sequence ID NO: amp-L AAT ACA TTCAAA TAT GTA TCC GC 54 amp-R TTA CCA ATG CTT AAT CAG TGA G 55 attB1-ACAAG TTTGTA CAAAAA AGCA GGCTAAT 56 ampL ACA TTC AAA TAT GTA TCC GCattB3- ACCAC TTTGTA CAAGAA AGCT GGGTTTA 57 ampR CCA ATG CTT AAT CAG TGAG attB1 + GGGG ACAAG TTTGTA CAAAAA AGCAGGCT 58 4-ampL AAT ACA TTC AAATAT GTA TCCGC attB3 + GGGG ACCAC TTTGTA CAAGAA AGCTGGGT 59 4-ampR TTACCA ATG CTT AAT CAG TGAG

[0391] PCR Amplification of Tet and Amp Gene Sequences

[0392] Six PCR reactions were performed, in 100 μl, consisting of 20 mMTris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM dNTPs, 2 ng linearizedpBR322, 2.5 units of Taq DNA polymerase (GIBCO-BRL), and 0.5 μM of eachpair of PCR primers listed in Tables 3 and 4. The reactions were firstheated to 94 C for 3′; then subjected to 25 cycles of 94° C. for 45seconds, 55° C. for 30 seconds, and 72° C. for 1 minute. Based on 1%agarose gel analysis, all the reactions generated products of theexpected size, in reasonable yields.

[0393] Purification of PCR Products

[0394] The products from duplicate reactions were pooled; extracted withan equal volume of phenol, isoamyl alcohol, and chloroform; extractedtwice with an equal volume of isobutanol; and precipitated with twovolumes of ethanol, as above. The six precipitates were washed once with100% ethanol, dried and dissolved in 100 μl TE. 1 μl aliquots were takenfor gel analysis of the product before PEG precipitation.

[0395] To each tube was added 50 μl of 30% PEG 8000, 30 mM MgCl₂. Thesolution was mixed well and centrifuged at 13,000 rpm for 10′, at roomtemperature. The supernatant was carefully removed, and the precipitatedissolved in 100 μl TE. Recovery was quantitated on a 1% agarose andestimated to be over 90%. The gel analysis also revealed that nucleicacid products smaller than about 300 nucleotides had been effectivelyremoved by the PEG precipitation step.

[0396] Recombination Reactions

[0397] Seven recombination reactions were performed, each in a totalvolume of 8 μl, containing 1× integrase buffer, 40 ng pEZC7102 (FIG.11A), and 2 μl recombinase mixture (see above, Example 10). Each of thereactions also contained approximately 40 ng of one of the six above PCRproducts or, as a positive control, 40 ng of pEZC7501 (FIG. 11B). Theamp and tet PCR products with attB sites at their termini are shown inFIGS. 12A and 12B. The reactions were incubated at 25° C. for 45′, at70° C. for 10′, then held at 0-5° C. for 1-2 hours until used totransform E. coli.

[0398]E. coli Transformation with Recombination Reaction Products

[0399] 1 μl of each of the recombination reactions was transformed intoMax Efficiency DH5α in a 50 μl transformation reaction, and followingexpression in S0C medium, ⅕ (100 μl) and ⅘ (400 μl) of each reactionwere plated on culture plates containing 50 μg/ml kanamycin. The plateswere incubated overnight and colonies were counted. The number ofcolonies obtained from each set of duplicate reactions are displayed inTable 13: TABLE 13 Recombination Reactions No. Colonies tet 100 (100 μl) 6, 10 tet 400 (400 μl) 27, 32 attB-tet 100  9, 6 attB-tet 400 27, 36attB + 4-tet 100 824, 1064 attB + 4-tet 400 >2000, >4000 amp 100  7, 13amp 400 59, 65 attB-amp 100 18, 22 attB-amp 400 66, 66 attB + 4-amp 1003020, 3540 attB + 4-amp 400 >5000, >5000 pEZC7501 100 320, 394 pEZC7501400 1188, 1400

[0400] Analysis of the Colonies Obtained

[0401] As a rapid phenotypic screen, 10 of the colonies from the tet EZCreactions and 33 of the colonies from the attB+4-tet EZC reactions werestreaked onto an LB culture plate containing tetracycline (15 μg/ml). Asa control for the potency of the tetracycline, 3 colonies ofpUC19-transformed cells, lacking a TetR gene, were also streaked ontothe plate. All colonies from the attB+4-tet EZC reactions grew well;colonies from the tet EZC reactions grew only very slightly, and thepUC19 colonies grew not at all.

[0402] Analogous results were obtained by streaking colonies from theamp PCR reactions on culture plates containing ampicillin (100 μg/ml ).All 21 colonies generated from the attB+4-amp recombination reactionsgrew well, whereas only one of 13 colonies from the attB-amp reactionsgrew in the presence of ampicillin. No growth was seen with any of the15 colonies from the recombination reaction with amp PCR products.

[0403] To characterize plasmid DNA, eight colonies generated from thesix EZC reactions with PCR products were picked into LB broth containing50 μg/ml kanamycin and grown overnight at 37° C. Miniprep DNA wasprepared from 0.9 ml of each culture, and the size of the supercoiledDNA was analyzed on a 1% agarose gel in TAE buffer containing 0.5 μg/mlethidium bromide. The results are displayed in Table 14. The predictedstructures of the recombination products are shown in FIGS. 12C and 12D.TABLE 14 Recombination Number with Reactions DNA Predicted Size (bp)Predicted Size tet SC 3386 0/8 (supercoiled) attB-tet SC 3386 1/8 attB +4-tet SC 3386 7/7 AvaI + Bam 485, 2901 3/3 amp SC 2931 0/8 attB-amp SC2931 3/8 attB + 4-amp SC 2931 8/8 Pst 429, 2502 3/3

[0404] Analysis

[0405] These results, based on the amplification of two different genesequences, tet and amp, within the plasmid pBR322 clearly demonstratethat PCR products generated using primers containing the 25 bp attB1 andattB3 recombination sequence serve as highly efficient substrates forthe recombination reaction. Addition of a short sequence to the outsideof each 25 bp attB site stimulates the recombination reaction by over100 fold, as also observed in the experiments of Example 10. Alsosimilar to Example 10, the efficiency of the recombination reactionsusing linear PCR products with attB sites exceeded the efficiencyobtained with the positive control SC DNA plasmid, pEZC7501.

[0406] Further, a high percentage of the reaction products are aspredicted, since all 33 colonies tested from the attB+4-tet reactionsdisplayed functional tetracycline resistance, and all 21 of the coloniesfrom the attB+4-amp reactions displayed ampicillin resistance. All 16 ofthe miniprep DNAs, examined from the recombination reactions of eitherattB+4-tet or attB+4-amp PCR products with pEZC7102, generatedsupercoiled DNA and restriction digest fragments of the correct sizes.

Example 12 Use of Topoisomerase to Stimulate Recombination

[0407] The stimulation of the recombination reaction by making one orthe parental plasmids linear was not expected. If the stimulationresulted from relief of some conformation constraint arising during thetwo recombination reactions (formation of the Cointegrate and resolutionto the two daughter molecules), then unwinding of the plasmids with atopoisomerase might also be stimulatory when one or both parentalplasmids were circular.

[0408] The Insert Donor was pEZC2901 (FIG. 7A), and the Vector Donor waspECZ3101 (FIG. 7B). A portion of pEZC3101 was linearized with Mlu I. 20ng of pEZC2901 and/or pECZ3101 were used in each 10 □l reaction (29 ngInt, 2.9 ng Xis, 5.4 ng IHF in 50 mM Tris HCl pH about 7.8, 16.5 mMNaCl, 35 mM KCl, 5 mM spermidine, 0.375 mg/ml BSA, 0.25 mM EDTA, 2%glycerol). Topoisomerase I (from calf Thymus; Life Technologes, Inc.)was diluted from 15 units/μl to the concentrations indicated in Table 15in 1× EZC buffer. TABLE 15 1 2 3 4 5 6 7 8 9 10 Circular 3101 2 2 2 2 2Linear 3101 2 2 2 2 2 Circular 2901 2 2 2 2 2 2 2 2 Recombinase 2 2 2 22 2 2 2 2 2 TE 2 2 Topoisomerase, 1:60 2 2 Topoisomerase, 1:20 2 2Topoisomerase, 1:6 2 2 3 × Buffer 2 2 2 2 2 2 2 2 2 2 1 × Buffer 2 2 2 2

[0409] These reactions were assembled in the following order: buffer;TE; DNAs; Clonase; Topoisomerase. The reactions were incubated at22°-28° for 45 minutes, then at 70° for 5 minutes. 1 μl aliquots weretransformed into UltraMax DH5α competent E. coli (Life Technologies,Inc.). Following expression, aliquots were plated on 100 μg/ml kanamycinand incubated at 30° for 48 hours. Results: see Table 16. TABLE 16 Re-Col- Insert action # onies Vector Donor Donor Recombinase Topoisomerase1 0 linear 3101 — + — 2 245 linear 3101 circular + — 2901 3 221 linear3101 circular + 0.5 units 2901 4 290 linear 3101 circular + 1.6 units2901 5 355 linear 3101 circular +   5 units 2901 6 0 circular 3101 + — 723 circular 3101 circular + — 2901 8 209 circular 3101 circular + 0.5units 2901 9 119 circular 3101 circular + 1.6 units 2901 10 195 circular3101 circular +   5 units 2901

[0410] Analysis

[0411] Linearizing the Vector Donor increased the number of coloniesabout 10 fold (reaction 2 vs. reaction 7). Addition of 0.5 to 5 units oftopoisomerase I to reactions containing circular Insert Donor and linearVector Donor had little or no effect on the number of colonies (reaction2 compared to reactions 3, 4, and 5; maximum 1.4 fold). In contrast, ifboth parental plasmids were circular (reaction 7-10), the addition oftopoisomerase stimulated the number of colonies 5 to 9 fold. Thusaddition of topoisomerase I to reactions in which both parental plasmidswere circular stimulated the recombination reactions nearly as much aslinearizing the Vector Donor parent. Topoisomerase I was active whenused in combination with the three recombination proteins, inrecombination buffer. The addition of topoisomerase I to therecombination reaction relieves the necessity to linearize the VectorDonor to achieve stimulation of the recombination reactions.

[0412] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be performed by modifying or changing the inventionwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any specificembodiment thereof, and that such modifications or changes are intendedto be encompassed within the scope of the appended claims.

[0413] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference.

1 60 1 25 DNA Unknown OTHER 18 “n” may be any nucleotide 1 rkycwgctttyktrtacnaa stsgb 25 2 25 DNA Unknown OTHER 18 “n” may be any nucleotide2 agccwgcttt yktrtacnaa ctsgb 25 3 25 DNA Unknown OTHER 18 “n” may beany nucleotide 3 gttcagcttt cktrtacnaa ctsgb 25 4 25 DNA Unknown OTHER18 “n” may be any nucleotide 4 agccwgcttt cktrtacnaa gtsgb 25 5 25 DNAUnknown OTHER 18 “n” may be any nucleotide 5 gttcagcttt yktrtacnaa gtsgb25 6 25 DNA Unknown Description of Unknown Organism recombinationproducts 6 agcctgcttt tttgtacaaa cttgt 25 7 25 DNA Unknown Descriptionof Unknown Organism recombination products 7 agcctgcttt cttgtacaaa cttgt25 8 25 DNA Unknown Description of Unknown Organism recombinationproducts 8 acccagcttt cttgtacaaa gtggt 25 9 25 DNA Unknown Descriptionof Unknown Organism recombination products 9 gttcagcttt tttgtacaaa cttgt25 10 25 DNA Unknown Description of Unknown Organism recombinationproducts 10 gttcagcttt cttgtacaaa cttgt 25 11 25 DNA Unknown Descriptionof Unknown Organism recombination products 11 gttcagcttt cttgtacaaagtggt 25 12 25 DNA Unknown Description of Unknown Organism recombinationproducts 12 agcctgcttt tttgtacaaa gttgg 25 13 25 DNA Unknown Descriptionof Unknown Organism recombination products 13 agcctgcttt cttgtacaaagttgg 25 14 25 DNA Unknown Description of Unknown Organism recombinationproducts 14 acccagcttt cttgtacaaa gttgg 25 15 25 DNA Unknown Descriptionof Unknown Organism recombination products 15 gttcagcttt tttgtacaaagttgg 25 16 25 DNA Unknown Description of Unknown Organism recombinationproducts 16 gttcagcttt cttgtacaaa gttgg 25 17 39 DNA Unknown Descriptionof Unknown Organism recombination products 17 ccaccacaaa cgcgtccatggaattacact ttaatttag 39 18 39 DNA Unknown Description of UnknownOrganism recombination products 18 ccaccacaag tcgacgcatg ccgacagccttccaaatgt 39 19 46 DNA Artificial Sequence Description of ArtificialSequence synthetic oligonucleotide 19 ggccgattac gatatcccaa cgaccgaaaacctgtatttt cagggt 46 20 30 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide 20 caggttttcg gtcgttgggatatcgtaatc 30 21 47 DNA Artificial Sequence Description of ArtificialSequence synthetic oligonucleotide 21 ggccagatta cgatatccca acgaccgaaaacctgtattt tcagggt 47 22 31 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide 22 caggttttcg gtcgttgggatatcgtaatc t 31 23 48 DNA Artificial Sequence Description of ArtificialSequence synthetic oligonucleotide 23 ggccaagatt acgatatccc aacgaccgaaaacctgtatt ttcagggt 48 24 32 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide 24 caggttttcg gtcgttgggatatcgtaatc tt 32 25 15 DNA Artificial Sequence Description of ArtificialSequence synthetic oligonucleotide 25 accgtttacg tggac 15 26 31 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide 26 tcgagtccac gtaaacggtt cccacttatt a 31 27 39 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide 27 uauuuucagg guatggagaa aaaaatcact ggatatacc 39 28 33DNA Artificial Sequence Description of Artificial Sequence syntheticoligonucleotide 28 ucccacuuau uacgccccgc cctgccactc atc 33 29 33 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide 29 uauuuucagg guatgcctgt tctggaaaac cgg 33 30 34 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide 30 ucccacuuau uatttcagcc ccagggcggc tttc 34 31 58 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide 31 tccgttgaag cctgcttttt tatactaact tgagcgaagcctcggggtca gcataagg 58 32 58 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide 32 ccaataactt cgtatagcatacattatacg aagttattgc cccttggtga catactcg 58 33 20 DNA ArtificialSequence Description of Artificial Sequence synthetic oligonucleotide 33tcactagtcg gcggcccaca 20 34 20 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide 34 gagcggcccc cgcggaccac20 35 21 DNA Artificial Sequence Description of Artificial Sequencesynthetic oligonucleotide 35 ggcccacaag tttgtacaaa a 21 36 20 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide 36 ccccgcggac cactttgtac 20 37 21 DNA ArtificialSequence Description of Artificial Sequence synthetic oligonucleotide 37acaagtttgt acaaaaaagc a 21 38 21 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide 38 accactttgt acaagaaagc t21 39 25 DNA Unknown Description of Unknown Organism recombinationproducts 39 rbycwgcttt yttrtacwaa stkgd 25 40 25 DNA Unknown Descriptionof Unknown Organism recombination products 40 asccwgcttt yttrtacwaastkgw 25 41 25 DNA Unknown Description of Unknown Organism recombinationproducts 41 asccwgcttt yttrtacwaa gttgg 25 42 25 DNA Unknown Descriptionof Unknown Organism recombination products 42 gttcagcttt yttrtacwaastkgw 25 43 25 DNA Unknown Description of Unknown Organism recombinationproducts 43 gttcagcttt yttrtacwaa gttgg 25 44 25 DNA Unknown Descriptionof Unknown Organism recombination products 44 tcggacgaaa aaatatgattgaact 25 45 25 DNA Unknown Description of Unknown Organism recombinationproducts 45 tcggacgaaa aaacatgttt gaaca 25 46 25 DNA Unknown Descriptionof Unknown Organism recombination products 46 tcggacgaaa gaacatgtttgaaca 25 47 25 DNA Unknown Description of Unknown Organism recombinationproducts 47 tgggtcgaaa gaacatgttt cacca 25 48 24 DNA Artificial SequenceDescription of Artificial Sequence synthetic oligonucleotide 48aattctcatg tttgacagct tatc 24 49 21 DNA Artificial Sequence Descriptionof Artificial Sequence synthetic oligonucleotide 49 cgatggatatgttctgccaa g 21 50 49 DNA Artificial Sequence Description of ArtificialSequence synthetic oligonucleotide 50 acaagtttgt acaaaaaagc aggctaattctcatgtttga cagcttatc 49 51 46 DNA Artificial Sequence Description ofArtificial Sequence synthetic oligonucleotide 51 accactttgt acaagaaagctgggtcgatg gatatgttct gccaag 46 52 53 DNA Artificial SequenceDescription of Artificial Sequence synthetic oligonucleotide 52ggggacaagt ttgtacaaaa aagcaggcta attctcatgt ttgacagctt atc 53 53 50 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide 53 ggggaccact ttgtacaaga aagctgggtc gatggatatgttctgccaag 50 54 23 DNA Artificial Sequence Description of ArtificialSequence synthetic oligonucleotide 54 aatacattca aatatgtatc cgc 23 55 22DNA Artificial Sequence Description of Artificial Sequence syntheticoligonucleotide 55 ttaccaatgc ttaatcagtg ag 22 56 48 DNA ArtificialSequence Description of Artificial Sequence synthetic oligonucleotide 56acaagtttgt acaaaaaagc aggctaatac attcaaatat gtatccgc 48 57 47 DNAArtificial Sequence Description of Artificial Sequence syntheticoligonucleotide 57 accactttgt acaagaaagc tgggtttacc aatgcttaat cagtgag47 58 52 DNA Artificial Sequence Description of Artificial Sequencesynthetic oligonucleotide 58 ggggacaagt ttgtacaaaa aagcaggcta atacattcaaatatgtatcc gc 52 59 51 DNA Artificial Sequence Description of ArtificialSequence synthetic oligonucleotide 59 ggggaccact ttgtacaaga aagctgggtttaccaatgct taatcagtga g 51 60 25 DNA Unknown Description of UnknownOrganism recombination products 60 agcctgcttt tttatactaa cttga 25

What is claimed is:
 1. A composition comprising: (a) an isolated nucleicacid molecule comprising one or more site-specific recombination sites;and (b) one or more topoisomerases bound to said isolated nucleic acidmolecule.
 2. The composition of claim 1, wherein said site-specificrecombination sites are att sites.
 3. The composition of claim 1,wherein said site-specific recombination sites are lox sites.
 4. Thecomposition of claim 3, wherein said lox sites are loxP sites.
 5. Thecomposition of claim 4, wherein said loxP sites are selected from thegroup consisting of loxP and loxP511.
 6. The composition of claim 2,wherein said att sites are selected from the group consisting of attB,attP, attL and attR.
 7. The composition of claim 6, wherein said attBsites are selected from the group consisting of attB1 (SEQ ID NO:6),attB2 (SEQ ID NO:7) and attB3 (SEQ ID NO:8).
 8. The composition of claim6, wherein said attP sites are selected from the group consisting ofattP1 (SEQ ID NO:15) and attP2 (SEQ ID NO:16).
 9. The composition ofclaim 6, wherein said attL sites are selected from the group consistingof attL1 (SEQ ID NO:12), attL2 (SEQ ID NO:13) and attL3 (SEQ ID NO:14).10. The composition of claim 6, wherein said attR sites are selectedfrom the group consisting of attR1 (SEQ ID NO:9), attR2 (SEQ ID NO:10)and attR3 (SEQ ID NO:11).
 11. A composition comprising: (a) an isolatednucleic acid molecule comprising two or more site-specific recombinationsites; and (b) one or more topoisomerases bound to said isolated nucleicacid molecule.
 12. The composition of claim 11, wherein said two or moresite-specific recombination sites do not recombine with each other. 13.An isolated nucleic acid molecule comprising: (a) one or moresite-specific recombination sites; and (b) one or more topoisomeraserecognition sites.
 14. The isolated nucleic acid molecule of claim 13,wherein said site-specific recombination sites are att sites areselected from the group consisting of attB, attP, attL and attR.
 15. Theisolated nucleic acid molecule of claim 13, wherein said site-specificrecombination sites are lox sites selected from the group consisting ofloxP and loxP511.
 16. The isolated nucleic acid molecule of claim 14,wherein said attB sites are selected from the group consisting of attB1(SEQ ID NO:6), attB2 (SEQ ID NO:7) and attB3 (SEQ ID NO:8).
 17. Theisolated nucleic acid molecule of claim 14, wherein said attP sites areselected from the group consisting of attP1 (SEQ ID NO:15) and attP2(SEQ ID NO:16).
 18. The isolated nucleic acid molecule of claim 14,wherein said attL sites are selected from the group consisting of attL1(SEQ ID NO:12), attL2 (SEQ ID NO:13) and attL3 (SEQ ID NO:14).
 19. Theisolated nucleic acid molecule of claim 14, wherein said attR sites areselected from the group consisting of attR1 (SEQ ID NO:9), attR2 (SEQ IDNO:10) and attR3 (SEQ ID NO:11).
 20. An isolated nucleic acid moleculecomprising: (a) two or more recombination sites that do not recombinewith each other; and (b) one or more topisomerase recognition sites.